Microbes and Metagenomics
in Human HealthAn overview of recent publications featuring Illumina® technology

TABLE OF CONTENTS
4

Introduction

5

Human Microbiome
Gut Microbiome
Gut Microbiome and Disease
Inflammatory Bowel Disease (IBD)
Metabolic Diseases: Diabetes and Obesity
Obesity
Oral Microbiome
Other Human Biomes

25

Viromes and Human Health
Viral Populations
Viral Zoonotic Reservoirs
DNA Viruses
RNA Viruses
Human Viral Pathogens
PhagesVirus Vaccine Development

44

Microbial Pathogenesis
Important Microorganisms in Human Health
Antimicrobial Resistance
Bacterial Vaccines

54

Microbial Populations
Amplicon Sequencing 16S: Ribosomal RNA
Metagenome Sequencing: Whole-Genome Shotgun Metagenomics
Eukaryotes
Single-Cell Sequencing (SCS)
Plasmidome
Transcriptome Sequencing

63

Glossary of Terms

64

Bibliography

This document highlights recent publications that demonstrate the use of Illumina technologies in immunology research. To learn
more about the platforms and assays cited, visit www.illumina.com.

An overview of recent publications featuring Illumina technology

3

INTRODUCTION
The study of microbes in human health traditionally focused on identifying andtreating pathogens in patients, usually with antibiotics. The rise of antibiotic

1.

Roca I., Akova M., Baquero F., Carlet J.,
Cavaleri M., et al. (2015) The global threat of
antimicrobial resistance: science for intervention. New Microbes New Infect 6: 22-29

2.

Shallcross L. J., Howard S. J., Fowler T. andDavies S. C. (2015) Tackling the threat of antimicrobial resistance: from policy to sustainableaction. Philos Trans R Soc Lond B Biol Sci
370: 20140082

3.

Sharland M., Saroey P. and Berezin E. N.
(2015) The global threat of antimicrobial resistance - The need for standardized surveillance
tools to define burden and develop interventions. J Pediatr (Rio J) 91: 410-4124.

National Institutes of H. (2012) NIH Human
Microbiome Project defines normal bacterial
makeup of the body. 2015: www.nih.gov/
news/health/jun2012/nhgri-13.htm; accessed
September 29, 2015

5.

Cho I. and Blaser M. J. (2012) The humanmicrobiome: at the interface of health anddisease. Nat Rev Genet 13: 260-270

6.

Handelsman J., Rondon M. R., Brady S.
F., Clardy J. and Goodman R. M. (1998)
Molecular biological access to the chemistryof unknown soil microbes: a new frontier fornatural products. Chem Biol 5: R245-249

7.

Franzosa E. A., Hsu T., Sirota-Madi A.,
Shafquat A., Abu-Ali G., et al. (2015) Sequencing and beyond: integrating molecular ‘omics’
for microbial community profiling. Nat Rev
Microbiol 13: 360-372

8.

Wade W. (2002) Unculturable bacteria—theuncharacterized organisms that cause oral
infections. Journal of the Royal Society of
Medicine 95: 81-83

9.

Peacock S. (2014) Health care: Bring microbial
sequencing to hospitals. Nature 509: 557-559

resistance and an increasingly dense—and mobile—global population is forcing achange in that paradigm.1, 2, 3 Improvements in high-throughput sequencing, also
called next-generation sequencing (NGS), allow a holistic approach to managing
microbes in human health.
In and on the human body, microbes outnumber human cells 10 to 1.4 Thesemicrobial cells constitute the human microbiome. Uncovering the members of thehuman microbiome, and their collective genes and functions, defines the role of our
microbial communities in health and disease. This goal can only be accomplished byhigh-throughput sequencing technologies paired with powerful analytic tools.5
Metagenomics is one of the fastest growing scientific disciplines, and it is becoming
a central tool for improving the quality of life worldwide. Originally conceived as thecollective genomes of nonculturable microorganisms,6 metagenomics now refers to
the use of high-throughput DNA sequencing to provide taxonomic (“Who is there?”)and functional (“What are they doing?”) profiles of microbial communities without
the need to culture the microbes in the laboratory.7 It is estimated that less than
2% of bacteria can be cultured in the laboratory. Our sudden ability to identify the8previously unstudied 98% of microbes—whether they are prokaryotes, eukaryotes,
or viruses—is revolutionizing and energizing the field of microbiology.
This document highlights recent publications that apply Illumina sequencing
technologies to metagenomics research.

Reviews“The application of sequencing technology to microbial
genomes will improve patient care and enhance publichealth. The feasibility and economics are clear.” – Peacock 201494

Microbes and metagenomics in human health

HUMAN MICROBIOME
The human microbiome encompasses the collection of microorganisms associatedwith the human body, including eukaryotes, archaea, bacteria and viruses.10 The
National Institutes of Health (NIH) Common Fund Human Microbiome Project
was established in 2008, with the mission of generating resources to enable thecomprehensive characterization of the human microbiome and analyze its role in healthand disease.11 This effort resulted in the characterization of microbial communities in10.

Human Microbiome Project C. (2012) A framework for human microbiome research. Nature
486: 215-221

11.

Foxman B. and Rosenthal M. (2013) Implications of the human microbiome project for
epidemiology. Am J Epidemiol 177: 197-201

12.

Morgan X. C., Segata N. and Huttenhower C.
(2013) Biodiversity and functional genomics
in the human microbiome. Trends Genet 29:
51-58

13.

Penders J., Stobberingh E. E., Savelkoul P. H.
and Wolffs P. F. (2013) The human microbiome
as a reservoir of antimicrobial resistance. Front
Microbiol 4: 87

different body parts, e.g., oral, gut, skin, and vaginal microbiomes. In parallel, research
has also characterized multiple biomes associated with disease states, highlighting theinfluence of microbiome dysbiosis in disease development.12 Some animal-associatedmicrobiomes have also been characterized in efforts to improve animal health andunderstand the interactions between humans and the environment.13

Reviews
DeWeerdt S. (2015) Microbiome: Microbial mystery. Nature 521: S10-11
Franzosa E. A., Hsu T., Sirota-Madi A., Shafquat A., Abu-Ali G., et al. (2015) Sequencing and beyond:integrating molecular ‘omics’ for microbial community profiling. Nat Rev Microbiol 13: 360-372
Koch L. (2015) Metagenomics: Shaping the gut microbiome. Nat Rev Genet 16: 2
Preidis G. A. and Hotez P. J. (2015) The newest “omics”--metagenomics and metabolomics--enter the battle
against the neglected tropical diseases. PLoS Negl Trop Dis 9: e0003382
Zhou J., He Z., Yang Y., Deng Y., Tringe S. G., et al. (2015) High-throughput metagenomic technologies forcomplex microbial community analysis: open and closed formats. MBio 6:
Fricke W. F. and Rasko D. A. (2014) Bacterial genome sequencing in the clinic: bioinformatic challenges andsolutions. Nat Rev Genet 15: 49-55
Lasken R. S. and McLean J. S. (2014) Recent advances in genomic DNA sequencing of microbial species
from single cells. Nat Rev Genet 15: 577-584
Peacock S. (2014) Health care: Bring microbial sequencing to hospitals. Nature 509: 557-559

References
Franzosa E. A., Morgan X. C., Segata N., Waldron L., Reyes J., et al. (2014) Relating themetatranscriptome and metagenome of the human gut. Proc Natl Acad Sci U S A 111: E2329-2338
The majority of microbiome studies require that samples be taken at specific facilities by trained personnel,
increasing costs and excluding some subjects from participation. The authors developed a protocol for
self-collection of stool and saliva samples, and frozen shipping within 24 hours of collection. They usedmetagenome and metatranscriptome analysis to assess the accuracy and feasibility of this protocol against
2 other sample handling methods. There was strong within-subject correlation between all 3 methods
at gene, species, and transcript levels, indicating that the developed protocol had minimal effect on
metagenomic profiles. Oral microbes co-occurring in stool samples displayed a drop in abundance andtranscript levels, suggesting that oral microbes that transit into the gut are not stable and do not play a rolein gut ecology. Overall, the study provides a robust method for self-collection and distinguishes relationships
between metatranscriptomes and metagenomes, highlighting the complementarity of both approaches in
microbiome studies.
Illumina Technology: HiSeq

An overview of recent publications featuring Illumina technology

5Lax S., Smith D. P., Hampton-Marcell J., Owens S. M., Handley K. M., et al. (2014) Longitudinal analysisof microbial interaction between humans and the indoor environment. Science 345: 1048-1052
Humans imprint their residences with their specific microbial communities. These homes are a good proxy
to monitor indoor microbial profiles related to their inhabitants. The Home Microbiome Project monitored
skin and home-surface microbial communities by 16S rRNA V4 amplicon sequencing. A total of 10
houses were monitored for 6 weeks, including 3 families before and after moving to a new home. Microbialcommunities were specific to each home/family pair because human inhabitants were the main source ofsuch microbiomes, which lead to acquisition of the family microbial community soon after a home move.
Genetically related individuals had insignificant microbiome differentiation, whereas a nonrelated individual
sharing common areas displayed some differentiation. Shotgun metagenomic sequencing revealed few
pathogens and their virulence genes on kitchen counter surfaces associated with specific house inhabitants.
This study highlights the interactions between humans and their environment and how this relationship may
reflect and impact health status.
Illumina Technology: Nextera XT DNA Library Prep Kit, HiSeq 2000, MiSeq

14.

Goodrich J. K., Waters J. L., Poole A. C.,
Sutter J. L., Koren O., et al. (2014) Human
genetics shape the gut microbiome. Cell 159:
789-799

15.

Dominguez-Bello M. G., Costello E. K.,
Contreras M., Magris M., Hidalgo G., et al.
(2010) Delivery mode shapes the acquisitionand structure of the initial microbiota acrossmultiple body habitats in newborns. Proc Natl
Acad Sci U S A 107: 11971-11975

16.

Veiga P., Pons N., Agrawal A., Oozeer R., Guyonnet D., et al. (2014) Changes of the humangut microbiome induced by a fermented milk
product. Sci Rep 4: 6328

17.

Wu G. D., Chen J., Hoffmann C., Bittinger
K., Chen Y. Y., et al. (2011) Linking long-term
dietary patterns with gut microbial enterotypes.
Science 334: 105-108

18.

Nakayama J., Watanabe K., Jiang J., Matsuda
K., Chao S. H., et al. (2015) Diversity in gut
bacterial community of school-age children in
Asia. Sci Rep 5: 8397

19.

Schnorr S. L., Candela M., Rampelli S.,
Centanni M., Consolandi C., et al. (2014) Gut
microbiome of the Hadza hunter-gatherers.
Nat Commun 5: 3654

20.

Yatsunenko T., Rey F. E., Manary M. J., TrehanI., Dominguez-Bello M. G., et al. (2012) Humangut microbiome viewed across age and geography. Nature 486: 222-227

21.

Lambert J. E., Myslicki J. P., Bomhof M. R.,
Belke D. D., Shearer J., et al. (2015) Exercise
training modifies gut microbiota in normal anddiabetic mice. Appl Physiol Nutr Metab 40:
749-752

22.

Erny D., Hrabe de Angelis A. L., Jaitin D.,
Wieghofer P., Staszewski O., et al. (2015) Host
microbiota constantly control maturation andfunction of microglia in the CNS. Nat Neurosci
18: 965-977

23.

Kamada N., Seo S. U., Chen G. Y. and Nunez
G. (2013) Role of the gut microbiota in immunity and inflammatory disease. Nat Rev Immunol
13: 321-335

24.

Kamada N. and Nunez G. (2013) Role of
the gut microbiota in the development andfunction of lymphoid cells. J Immunol 190:
1389-1395

25.

Markle J. G., Mortin-Toth S., Wong A. S.,
Geng L., Hayday A., et al. (2013) gammadelta
T cells are essential effectors of type 1 diabetes in the nonobese diabetic mouse model. J
Immunol 190: 5392-5401

26.

Org E., Mehrabian M. and Lusis A. J. (2015)
Unraveling the environmental and genetic interactions in atherosclerosis: Central role of thegut microbiota. Atherosclerosis 241: 387-399

27.

Kim D. H. (2015) Gut Microbiota-MediatedDrug-Antibiotic Interactions. Drug Metab
Dispos 43: 1581-1589

28.

Suez J., Korem T., Zeevi D., Zilberman-Schapira G., Thaiss C. A., et al. (2014) Artificial
sweeteners induce glucose intolerance by altering the gut microbiota. Nature 514: 181-186

29.

Feng Q., Liang S., Jia H., Stadlmayr A., Tang
L., et al. (2015) Gut microbiome development
along the colorectal adenoma-carcinoma
sequence. Nat Commun 6: 6528

30.

Nayfach S., Fischbach M. A. and Pollard
K. S. (2015) MetaQuery: a web server for
rapid annotation and quantitative analysis of
specific genes in the human gut microbiome.
Bioinformatics

Gut Microbiome
In the gut, the host provides whole foods while the microbiota makes nutrients available
to the host. This symbiotic relationship also keeps opportunistic pathogens at bay.
Microbiota composition is variable among individuals due to the influence of multiple
factors, including genetics,14 birth mode,15 diet,16, 17 geographic region,18, 19 age,20 andeven exercise.21 This complex microbial biome influences many biological aspects ofhuman health, such as the immune system,22, 23, 24 diabetes,25 atherosclerosis,26 and theinteraction with xenobiotics.

27

In the gut, the host provides whole foods while the microbiota makes nutrients available to the host.

Diet and modern processed foods can alter microbiomes and metabolic pathways in
unforeseen ways. For example, certain artificial sweeteners reduce glucose tolerance
in mice through intestinal dysbiosis.28 Significant shifts in microbiota composition canalso occur before or during the development of colorectal cancer in humans. These
shifts could be potential biomarkers for diagnostics and disease management, andthey can place emphasis on gut microbiome characterization.29 Bioinformatic tools
have been developed to identify bacterial species, genes/pathways, and to support
inferences associated with clinical variables.30

6Microbes and metagenomics in human healthReviews
Dietert R. R. and Silbergeld E. K. (2015) Biomarkers for the 21st century: listening to the microbiome. Toxicol
Sci 144: 208-216
Gritz E. C. and Bhandari V. (2015) The human neonatal gut microbiome: a brief review. Front Pediatr 3: 17
Koch L. (2015) Metagenomics: Shaping the gut microbiome. Nat Rev Genet 16: 2
Candela M., Turroni S., Biagi E., Carbonero F., Rampelli S., et al. (2014) Inflammation and colorectal cancer,
when microbiota-host mutualism breaks. World J Gastroenterol 20: 908-922
Cenit M. C., Matzaraki V., Tigchelaar E. F. and Zhernakova A. (2014) Rapidly expanding knowledge on therole of the gut microbiome in health and disease. Biochim Biophys Acta 1842: 1981-1992
Salazar N., Arboleya S., Valdes L., Stanton C., Ross P., et al. (2014) The human intestinal microbiome atextreme ages of life. Dietary intervention as a way to counteract alterations. Front Genet 5: 406
Walker A. W., Duncan S. H., Louis P. and Flint H. J. (2014) Phylogeny, culturing, and metagenomics of thehuman gut microbiota. Trends Microbiol 22: 267-274

References
Feng Q., Liang S., Jia H., Stadlmayr A., Tang L., et al. (2015) Gut microbiome development along thecolorectal adenoma-carcinoma sequence. Nat Commun 6: 6528
Benign polyps slowly turn to colorectal cancer, but changes in gut microbiota during progression of disease are notwell understood. Metagenome-wide association studies on stool samples from adenoma and carcinoma patientsand healthy controls were used to assess microbes, genes and functions enriched in each group. Profoundshifts in gut microbiota associated with tumors were observed, such as an increase of specific Bacillus species
in progression from healthy to advanced carcinomas. The authors identified specific gene markers enriched in
carcinoma samples as potential biomarkers of disease. A possible risk factor for disease is higher consumptionof red meat relative to vegetables, due to the outgrowth of bacteria hostile to the gut environment promoted byamino acid degradation in the colon. Likewise, lactic acid producers enriched in control samples might inhibit
amino acid degradation in the colon and inhibit potential pathogens, contributing to a nurturing gut environment.
Illumina Technology: HiSeq 100 bp paired-end (PE) reads

Intestinal polyps.
Martinez I., Stegen J. C., Maldonado-Gomez M. X., Eren A. M., Siba P. M., et al. (2015) The gut
microbiota of rural papua new guineans: composition, diversity patterns, and ecological processes.
Cell Rep 11: 527-538
The gut microbiota is influenced by factors such as lifestyle and diet; alterations in these factors may lead tohealth disturbances. Amplicon sequencing of 16S V5 rRNA was used to assess the community differences infecal samples from rural Papua New Guinea (PNG, nonindustrialized) and US (Westernized) residents. PNG
and US residents shared a core of the most abundant phylotypes, although there were substantial differencesin the specific microbiome signatures for each population. PNG microbiota was characterized by bacterial
homogenizing dispersal processes that resulted in lower interindividual variation, greater bacterial diversity
and abundance, and the presence of bacterial species unique to this population. The authors suggest thatmicrobiome differences may result from population heterogeneity and hygiene practices used in Westernizedsocieties that limit bacterial dispersal processes found in nonindustrialized microbiomes. It is likely that a
combination of factors contributes to the observed differences.
Illumina Technology: MiSeq

An overview of recent publications featuring Illumina technology

7Papua New Guineans harbor a characteristically diverse gut microbiota that distinguishes them from themicrobiota found in industrialized societies.
Obregon-Tito A. J., Tito R. Y., Metcalf J., Sankaranarayanan K., Clemente J. C., et al. (2015)
Subsistence strategies in traditional societies distinguish gut microbiomes. Nat Commun 6: 6505
Amplicon sequencing of 16S V4 rRNA gene and metagenome sequences were used to compare two
traditional populations from Peru (hunter-gatherers and an agricultural community) and a typical US
community. Both traditional communities had higher microbial diversity than the US cohort. Available datafrom similar populations in distant geographic locations support these findings, showing clear separation
between traditional and industrial microbiomes. Rural communities were enriched for nonpathogenic
Treponema similar to the carbohydrate metabolizer T. succinifaciens. Similar reports in ancient microbiomes
and other traditional populations suggest that these microbes may have been lost through Westernization but
remain in traditional populations due to their potential metabolic functions.
Illumina Technology: Nextera DNA Library Prep Kit, HiSeq v4
Arthur J. C., Gharaibeh R. Z., Muhlbauer M., Perez-Chanona E., Uronis J. M., et al. (2014) Microbial genomic analysis
reveals the essential role of inflammation in bacteria-induced colorectal cancer. Nat Commun 5: 4724
The complex relationships among microbiota, inflammation, and pathology in colorectal cancer are still
undefined. 16S V6 rRNA gene amplicon sequencing and RNA-Seq were used to define gut microbiota
composition in a colitis model using ex-germ-free interleukin 10-/- mice. The authors showed that
inflammation was essential for development of E. coli–induced colorectal cancer in an inflammation-sensitive
genetic background. This model illustrates how gut environment can alter microbial gene expression and how
inflammation is a defining factor in E. coli–induced cancer.
Illumina Technology: TruSeq RNA Library Prep Kit v2, HiSeq 2000
Goodrich J. K., Waters J. L., Poole A. C., Sutter J. L., Koren O., et al. (2014) Human genetics shape
the gut microbiome. Cell 159: 789-799
The interplay of host genetics, gut microbiota, and metabolic patterns can impact disease and obesity.
Defining this interrelationship could open new avenues for disease prevention, diagnosis, and management.
Self-collected fecal samples from 416 adult twin pairs were subjected to amplicon sequencing of 16S
V4 rRNA genes. The abundance of specific microbiota members was influenced by host genetics: taxa
abundance was more correlated in monozygotic than dizygotic twin pairs. The authors found highly heritablebacterial taxa (Christensenellaceae) forming co-occurrence networks with other heritable bacteria andmethanogenic archaea. These networks were enriched in individuals with low body mass. Introducing
Christensenellaceae minuta to obese microbiomes followed by transplantation to germ-free mice reduced
weight gain in the recipients, who acquired stable changes in community diversity.
Illumina Technology: MiSeq 250 bp PE reads
Hsiao A., Ahmed A. M., Subramanian S., Griffin N. W., Drewry L. L., et al. (2014) Members of thehuman gut microbiota involved in recovery from Vibrio cholerae infection. Nature 515: 423-426
Changes in gut microbiota during or after diarrhea episodes, caused by infectious agents, are not wellcharacterized. As a result, their contribution to recovery or susceptibility remains poorly understood. The
results from amplicon sequencing of 16S V4 rRNA gene and metagenomic analysis of fecal samples from
Bangladeshi adults during and recovering from V. cholerae diarrhea were examined. Microbial composition

8

Microbes and metagenomics in human healthduring recovery parallels the establishment patterns found in healthy Bangladeshi children. Fecal transplantof the recovering microbiota into gnotobiotic mice revealed that Ruminococcus oleum, associated with infant
microbiota establishment and recovery from V. cholerae diarrhea, was able to restrict V. cholerae colonization
by a quorum-sensing autoinducer-2 mechanism and a novel regulatory pathway. The authors highlight thevalue of mining microbiota data to identify mechanisms that limit colonization by enteropathogens.

31.

Sack D. A., Sack R. B., Nair G. B. andSiddique A. K. (2004) Cholera. Lancet 363:
223-233

Illumina Technology: MiSeq 250 bp PE reads, Genome AnalyzerIIx, HiSeq 2000

Cholera has been prevalent in the Ganges delta since ancient times.31
Integrative H. M. P. R. N. C. (2014) The Integrative Human Microbiome Project: dynamic analysis of
microbiome-host omics profiles during periods of human health and disease. Cell Host Microbe 16:276-289
The Human Microbiome Project (HMP) generated a wealth of protocols, bioinformatic pipelines, and data. The
Integrative HMP (iHMP) Research Network Consortium will carry out the second phase of the HMP, aiming
to understand microbiome-host interactions by monitoring microbiome and host changes for a period of 3
years. Phylogenetic composition (amplicon and shotgun sequencing) and functional data from multiple ‘omic
approaches (transcriptome, proteome) will be generated from 3 longitudinal studies with focus on: i) pregnancy;ii) gut disease onset (inflammatory bowel disease (IBD) model); and iii) respiratory viral infection and onset oftype 2 diabetes.
Illumina Technology: Illumina platforms will be used in the study
Li J., Jia H., Cai X., Zhong H., Feng Q., et al. (2014) An integrated catalog of reference genes in thehuman gut microbiome. Nat Biotechnol 32: 834-841
Multiple gut microbiome studies have generated large gene datasets. Unifying data to generate a global
representative catalog of reference genes would allow for integrative study of the gut microbiome. A
nonredundant catalog comprised of 9,879,896 human gut microbial genes was generated by combining
249 newly sequenced samples, 1018 samples from MetaHit, HMP and a diabetes study, as well as
511 sequenced genomes of gut bacteria and archaea. The integrated gene catalog (IGC) was tested bysuccessful mapping and correlation of metatranscriptomic and metagenomic data from a recent study.
IGC was used to demonstrate country-specific gut microbiome signatures in Chinese and Danish cohorts,
highlighting its usefulness to facilitate characterization of variation across populations.
Illumina Technology: Genome AnalyzerIIx and available Illumina-generated data from previous studies
Moeller A. H., Li Y., Mpoudi Ngole E., Ahuka-Mundeke S., Lonsdorf E. V., et al. (2014) Rapid changes
in the gut microbiome during human evolution. Proc Natl Acad Sci U S A 111: 16431-16435
The evolutionary history of the human gut microbiome can be elucidated by studying the microbiomes of
nonhuman primates. Amplicon sequencing of the 16S V4 rRNA gene was used to characterize the gut
microbiomes of diverse African apes (chimpanzees, bonobos, and gorillas). This information was used togenerate a model for evolutionary divergence of the current human microbiome from ancestral populations
through reconstruction of microbiome changes. African ape gut microbiomes followed slow compositionalchanges during diversification, but remain highly diverse in comparison to humans. The authors propose thathumans have followed accelerated rates of divergence, characterized by the loss of microbial diversity as an
adaptation to animal-based diets.
Illumina Technology: MiSeq

An overview of recent publications featuring Illumina technology

9

Mountain gorilla (Gorilla gorilla berengeii) eating leaves, Park du Volcanes, Rwanda. African apes havebeen used as models of gut microbiome changes during human evolution.
Rosser E. C., Oleinika K., Tonon S., Doyle R., Bosma A., et al. (2014) Regulatory B cells are induced by
gut microbiota-driven interleukin-1beta and interleukin-6 production. Nat Med 20: 1334-1339
Regulatory B (Breg) cells prevent excessive immune responses by release of interleukin (IL)-10. These cells
differentiate upon inflammation, but the spectrum of signals involved in this process is unknown. The
authors treated mice with antibiotics to deplete their gut microbiota. Antigen-induced arthritis of these mice
resulted in milder arthritis and reduced numbers of immune cells and proinflammatory cytokines. Similar
disturbances were found in splenocytes and mesenteric lymph nodes, along with reduced function anddifferentiation of Breg cells. In the arthritis-induced model, proinflammatory cytokines IL-1β and IL-6 were
produced by conventionally housed mice but not in sterile-housed animals. These cytokines promote Bregcell differentiation and IL-10 production, and their signals are produced in response to gut microbiota andarthritis. The authors suggest that IL-1β, IL-6, and th17 differentiation are signals also required for Breg celldifferentiation. In addition, a small variation in commensal microbiota contributes to both proinflammatory andregulatory responses behind these dynamics.
Illumina Technology: MiSeq 250 bp PE reads
Schwab C., Berry D., Rauch I., Rennisch I., Ramesmayer J., et al. (2014) Longitudinal study of murine
microbiota activity and interactions with the host during acute inflammation and recovery. ISME J 8:
1101-1114
Gut microbiota functional changes and recovery patterns after colitis have not been addressed in detail.
Metatranscriptomics, 16S rRNA amplicon sequencing, and host markers were used to monitor changes
in gut microbiota during acute inflammation and recovery in a mouse model of colitis induced by dextran
sodium sulfate. Severe dysbiosis was observed during acute colitis, followed by gradual recovery of theoriginal microbiota composition. However, microbial gene expression was more resilient to disturbance andseemed to recover quickly. Host inflammation markers correlated with acute colitis, reduced transcripts
of commensal clostridial flagellar genes, and an increase in transcripts related to mucin degradation. The
authors propose that the gut microbiota has a remarkable ability to recover and inflammation triggers immune
reactions against flagella of commensal bacteria that results in temporary microbiota dysbiosis.
Illumina Technology: HiSeq
Suez J., Korem T., Zeevi D., Zilberman-Schapira G., Thaiss C. A., et al. (2014) Artificial sweeteners
induce glucose intolerance by altering the gut microbiota. Nature 514: 181-186
Noncaloric artificial sweeteners (NAS) are widely used due to their low caloric content; most of these pass
the gastrointestinal tract undigested by the host and directly encounter the intestinal microbiota. Ampliconsequencing of 16S V2 rRNA and shotgun metagenomics were used in fecal samples of a small human cohortand mice fed different NAS. Mice developed glucose intolerance after chronic exposure to NAS. Humans
developed poor glycemic responses with gut microbiota compositional changes. Glucose tolerance wasreinstituted by fecal transplant and abolished by antimicrobial treatment. A shift in microbial composition andabundance further corroborated the contribution of gut dysbiosis in NAS-induced glucose tolerance. Glycan
degradation pathways were overrepresented in NAS-fed mice, which likely resulted in increased energy
harvest and production of precursors of de novo glucose and lipid synthesis. The authors stress the need toreconsider the effects of NAS in humans.
Illumina Technology: MiSeq, HiSeq

10

Microbes and metagenomics in human healthWang J., Linnenbrink M., Kunzel S., Fernandes R., Nadeau M. J., et al. (2014) Dietary history
contributes to enterotype-like clustering and functional metagenomic content in the intestinal
microbiome of wild mice. Proc Natl Acad Sci U S A 111: E2703-2710
The existence of enterotypes characterized by specific gut microbiota signature taxa is still a topic of debate.
Amplicon sequencing of 16S V1 and V2 rRNA gene, shotgun metagenomics, and diet reconstruction wereused to assess enterotypes in wild house mice. The authors identified 2 clusters similar to those identified inhumans and other mammals. The clusters displayed differences in gene transcripts involved in carbohydrate
and protein metabolism pathways that relate to plant and meat food sources. However, enterotype
classification changed quickly upon diet change. In addition, changes associated with different environmental
settings and reconstruction of dietary history in wild-caught mice revealed that enterotype-like clustering was
highly influenced by diet.

32.

Chen Y., Zhao Y., Cheng Q., Wu D. and Liu
H. (2015) The Role of Intestinal Microbiota in
Acute Graft-versus-Host Disease. J Immunol
Res 2015: 145859

33.

Zanella Terrier M. C., Simonet M. L., Bichard P.
and Frossard J. L. (2014) Recurrent Clostridium difficile infections: the importance of theintestinal microbiota. World J Gastroenterol 20:
7416-7423

34.

David L. A., Weil A., Ryan E. T., Calderwood
S. B., Harris J. B., et al. (2015) Gut microbial
succession follows acute secretory diarrhea inhumans. MBio 6: e00381-00315

35.

Singh R., Nieuwdorp M., ten Berge I. J.,
Bemelman F. J. and Geerlings S. E. (2014) The
potential beneficial role of faecal microbiotatransplantation in diseases other than Clostridium difficile infection. Clin Microbiol Infect 20:
1119-1125

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Wikoff W. R., Anfora A. T., Liu J., Schultz P.
G., Lesley S. A., et al. (2009) Metabolomics
analysis reveals large effects of gut microflora
on mammalian blood metabolites. Proc Natl
Acad Sci U S A 106: 3698-3703

37.

Rath C. M. and Dorrestein P. C. (2012) The
bacterial chemical repertoire mediates metabolic exchange within gut microbiomes. Curr
Opin Microbiol 15: 147-154

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Jacobsen U. P., Nielsen H. B., HildebrandF., Raes J., Sicheritz-Ponten T., et al. (2013)
The chemical interactome space between thehuman host and the genetically defined gut
metabotypes. ISME J 7: 730-742

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Jia W., Li H., Zhao L. and Nicholson J. K.
(2008) Gut microbiota: a potential new territory
for drug targeting. Nat Rev Drug Discov 7:
123-129

Illumina Technology: HiSeq 2000 100 bp PE reads

Gut Microbiome and Disease
Several recent studies link alterations of the individual microbiome (or dysbiosis) withdifferent diseases of clinical importance. From chronic inflammation to infectious
diseases, the gut microbiome has an expanding role in health and disease, including
a new possible role in acute graft-versus-host disease.32
Restoration of gut microbiota is a promising alternative therapy that addresses
intestinal dysbiosis, mimicking normal microbial succession after intestinal events.34
33

Clostridium difficile colitis, a recurring leading cause of nosocomial-associatedinfections, is a well-characterized model of antibiotic-related intestinal dysbiosis.
The dysbiosis is characterized by reduced taxa diversity that results in uncontrolled
proliferation of C. difficile. Successful microbiota restoration can reduce therecurrence of C. difficile.35
The development of diagnostic tests associated with specific microbiome signatures,customized therapeutics, development of new antimicrobials, targeted diets, and the
profiling of individual biomes might become future tools for personalized health care.
A new generation of nontoxic small molecules, identified by analysis of combined
omics approaches, can interfere with pathways or metabolites associated withdisease.36, 37, 38, 39

Reviews
Gritz E. C. and Bhandari V. (2015) The human neonatal gut microbiome: a brief review. Front Pediatr 3: 17
Shono Y., Docampo M. D., Peled J. U., Perobelli S. M. and Jenq R. R. (2015) Intestinal microbiota-related
effects on graft-versus-host disease. Int J Hematol 101: 428-437

An overview of recent publications featuring Illumina technology

11References
Buffie C. G., Bucci V., Stein R. R., McKenney P. T., Ling L., et al. (2015) Precision microbiome
reconstitution restores bile acid mediated resistance to Clostridium difficile. Nature 517: 205-208
Clostridium difficile is a major cause of antibiotic-induced diarrhea in hospitalized patients. However, thebacteria involved in sensitivity or resistance to C. difficile colonization are not known. Amplicon sequencingof the 16S V4-V5 rRNA gene was used in conjunction with targeted depletion of gut microbiota to evaluate
the role of different bacterial populations in C. difficile colonization in mice. C. scindens, a bile secondaryacid synthesis species common to humans and mice, was associated with resistance to C. difficile infection.
Enhanced resistance was achieved upon addition of human-derived C. scindens in mice and correlated withsynthesis of secondary bile acids, which corroborated a functional role in C. difficile inhibition. This approach
identified single bacteria, out of the rich intestinal microbiota context, that were able to inhibit C. difficile. Its
potential use in fecal transplants, microbiota manipulation, and an initial antimicrobial approach is consideredin light of the biological aspects inherent to complex microbiota interactions.
Illumina Technology: TruSeq DNA Library Prep Kit, MiSeq
He B., Nohara K., Ajami N. J., Michalek R. D., Tian X., et al. (2015) Transmissible microbial andmetabolomic remodeling by soluble dietary fiber improves metabolic homeostasis. Sci Rep 5: 10604
Dietary management can help improve metabolic diseases by promoting health benefits not yet totally
characterized. Digestion-resistant plant-derived fibers, like maltodextrin (RM), improve glucose and lipid
homeostasis and help reduce weight gain. The authors fed RM to obese mice, tested its effects in glucose
tolerance, and monitored gut microbiota changes by amplicon sequencing of the 16S V4 rRNA gene. RM
improved glycemic control by decreasing fasting glucose levels. RM induced beneficial gut microbiota
remodeling by increasing the abundance of beneficial bacteria (Lactobacillus and Bifidobacterium) anddecreasing fat-associated bacteria (Alistipes). Fecal transplantation corroborated the positive effects of RMremodeled gut microbiota, which was accompanied by metabolic changes, such as improved cholesterol andglucose metabolism. The study sheds light on the mechanisms underlying the beneficial effects of RM.
Illumina Technology: MiSeq 250 bp PE reads, CASAVA v1.8.3
Weingarden A., Gonzalez A., Vazquez-Baeza Y., Weiss S., Humphry G., et al. (2015) Dynamic changes
in short- and long-term bacterial composition following fecal microbiota transplantation for recurrent
Clostridium difficile infection. Microbiome 3: 10
Fecal transplants can restore the gut microbiome in patients suffering recurring C. difficile infections
refractory to antimicrobial therapy. The stability and long-lasting effects of fecal transplantation have not beenaddressed, despite its increasing use. Amplicon sequencing of the 16S rRNA gene was used to monitor
gut microbiota changes in patients with C. difficile treated by fecal transplantation for up to 151 days after
treatment. Microbial composition shifted from severe dysbiosis to microbiomes similar to the healthy cohort
established in the HMP. Upon fecal transplant, the recipient gut microbiota resembled that from the donor but
diverged over time in each treated individual. The authors showed a very dynamic picture of variable microbialchanges that succeeded each other over time and by individual. This surveillance can only be achieved byfrequent sampling and close monitoring of gut microbiomes using high-throughput methods.
Illumina Technology: MiSeq v3 150 bp PE reads
Yin J., M P., Wang S., Liao S. X., Peng X., et al. (2015) Different Dynamic Patterns of beta-Lactams,
Quinolones, Glycopeptides and Macrolides on Mouse Gut Microbial Diversity. PLoS One 10: e0126712
The use of oral antimicrobials is a common practice in hospital and nonhospital settings. These compounds
come into contact with gut microbiota and have recognizable effects, although fine detail about specificdisturbances is still needed. Amplicon sequencing of the 16S V6 rRNA gene was used to monitor gut
microbial changes caused by administration of 6 commonly used antimicrobials in BALB/c mice. Ceftriaxone
sodium, cefoperazone/sulbactam, meropenem and vancomycin resulted in quick dramatic changes.
Azithromycin caused detectable changes, and ofloxacine effect was minimal. β-lactams inhibited Alistipes
and favored the growth of Clostridium, which may influence the development of C. difficile infections after
β-lactam use. In contrast, vancomycin favored E. coli growth and inhibited Clostridium. Lastly, azithromycin
and meropenem induced the growth of Enterococcus. This study sheds light on the effects of commonlyused antibiotics in gut microbiota, which can take place swiftly and have long-lasting effects in the
microbial community.
Illumina Technology: TruSeq DNA Library Prep Kit, HiSeq 2000 100 bp PE reads

12

Microbes and metagenomics in human healthLukens J. R., Gurung P., Vogel P., Johnson G. R., Carter R. A., et al. (2014) Dietary modulation of the
microbiome affects autoinflammatory disease. Nature 516: 246-249
Inflammatory diseases have been on the rise for a few years, and there is evidence that changes in dietary
habits may have a role in disease development. In this study, the authors used a mouse osteomyelitis
model (Pstpip2cmo) and amplicon sequencing of the 16S V4 rRNA gene to assess the contribution of diet to
inflammatory disease. Mice with osteomyelitis had enriched abundance of Prevotella. Diet-induced protection
against osteomyelitis reduced expression of pro-IL-1β in distant neutrophils. The study shows that dietchanges can limit inflammation in a mouse osteomyelitis model and thus contribute to the interplay between
inflammatory disease and gut microbiota.
Illumina Technology: MiSeq
Morgan A. P., Crowley J. J., Nonneman R. J., Quackenbush C. R., Miller C. N., et al. (2014) The
antipsychotic olanzapine interacts with the gut microbiome to cause weight gain in mouse.
PLoS One 9: e115225
Olanzapine is prescribed as treatment for schizophrenia, bipolar disorder, and other disorders. However,patients receiving this medication exhibit dramatic weight gain by a yet unknown mechanism. Ampliconsequencing of the 16S v4 rRNA gene was used to survey microbiota changes in mice receiving olanzapine(crossover experimental design). The drug potentiated variable weight gain in 8 different inbred mice strains
under a high-fat diet (HFD). This effect was associated to gut microbiota, as demonstrated by crossoverexperiments where olanzapine and a HFD enriched “obesogenic” bacteria in the gut microbiota of mice.
Furthermore, olanzapine was able to impair or delay growth of 2 abundant commensal enteric bacteria in
vitro. The study shows 3 pieces of evidence that strongly suggest an obesogenic effect of olanzapine byaltering gut microbiota composition. It pinpoints the importance of gut microbiota as a biomarker and as apotential therapeutic target in drug-induced weight gain.
Illumina Technology: Nextera DNA Library Prep Kit, MiSeq 250 bp PE reads, CASAVA v1.8.2
Stefka A. T., Feehley T., Tripathi P., Qiu J., McCoy K., et al. (2014) Commensal bacteria protect against
food allergen sensitization. Proc Natl Acad Sci U S A 111: 13145-13150
Food allergy may be exacerbated by changes in commensal microbiota, although specific mechanisms orprotective/sensitive bacterial taxa have not been addressed in detail. Amplicon sequencing of the 16S V4rRNA gene and selective colonization of gnotobiotic mice identified a bacterial community enriched with
Clostridia as allergy-protective. This protective Clostridia induced novel regulatory mechanisms of innate
lymphoid cell function related to activation of Treg cells, IgA production in the colon, and regulation of allergenaccess to the bloodstream. Additionally, intestinal epithelial permeability was upregulated by Clostridia, andantimicrobial depletion of the neonatal gut microbiota resulted in increased sensitization to food allergens.
These findings collectively: i) suggest a role for gut microbiota in food allergy; ii) identify a “protective”
community; and iii) raise the possibility of microbiota manipulation to prevent or treat food allergies.
Illumina Technology: Illumina MouseRef-8 array, MiSeq

Gnotobiotic mice are used to determine the role of microbes in food allergen sensitization.

An overview of recent publications featuring Illumina technology

13

Inflammatory Bowel Disease (IBD)The MetaHit project aimed to characterize the relationship between microbial genes
and intestinal health and disease, with a focus on IBD and obesity.40 In general,

40.

Lee J. S., Rhee J. S., Kim R. O., Hwang D. S.,
Han J., et al. (2010) The copepod Tigriopus
japonicus genomic DNA information (574Mb)
and molecular anatomy. Mar Environ Res 69
Suppl: S21-23

41.

Cotillard A., Kennedy S. P., Kong L. C., Prifti
E., Pons N., et al. (2013) Dietary intervention
impact on gut microbial gene richness. Nature
500: 585-588

42.

Bindels L. B., Neyrinck A. M., Salazar N.,
Taminiau B., Druart C., et al. (2015) Non
Digestible Oligosaccharides Modulate the
Gut Microbiota to Control the Development of
Leukemia and Associated Cachexia in Mice.
PLoS One 10: e0131009

43.

Greenblum S., Carr R. and Borenstein E.
(2015) Extensive strain-level copy-number variation across human gut microbiome species.
Cell 160: 583-594

44.

Wang W., Jovel J., Halloran B., Wine E., Patterson J., et al. (2015) Metagenomic analysisof microbiome in colon tissue from subjects
with inflammatory bowel diseases reveals interplay of viruses and bacteria. Inflamm Bowel
Dis 21: 1419-1427

45.

Cammarota G., Ianiro G., Cianci R., Bibbo S.,
Gasbarrini A., et al. (2015) The involvement ofgut microbiota in inflammatory bowel disease
pathogenesis: potential for therapy. Pharmacol
Ther 149: 191-212

low diversity is a dysbiosis often found in chronic inflammatory diseases and other
health disturbances, a defect that might be controlled to some extent by dietary
management.41 Dietary management could also alleviate the intestinal symptoms
associated with cancer.42
Metagenomic analysis has linked variation in microbial gene copy number, at thestrain level, to IBD and obesity.43 Viral sequences have also been recovered frombiopsy samples, suggesting an interplay between viral and bacterial elements.44 This
disturbance plays a role in the immunopathogenesis of IBD, given the reciprocal
relationship between the development and performance of the immune system andits associated gut microbiome.45 The complexity of IBD and the importance of ahealthy microbiome demonstrate the need to address gut microbiome management
as a reasonable measure to control this and other diseases.

Reviews
Cammarota G., Ianiro G., Cianci R., Bibbo S., Gasbarrini A., et al. (2015) The involvement of gut microbiota ininflammatory bowel disease pathogenesis: potential for therapy. Pharmacol Ther 149: 191-212
Ohman L., Tornblom H. and Simren M. (2015) Crosstalk at the mucosal border: importance of the gut
microenvironment in IBS. Nat Rev Gastroenterol Hepatol 12: 36-49
Bringiotti R., Ierardi E., Lovero R., Losurdo G., Di Leo A., et al. (2014) Intestinal microbiota: The explosive
mixture at the origin of inflammatory bowel disease? World J Gastrointest Pathophysiol 5: 550-559
Ferreira C. M., Vieira A. T., Vinolo M. A., Oliveira F. A., Curi R., et al. (2014) The central role of the gut
microbiota in chronic inflammatory diseases. J Immunol Res 2014: 689492
Gkouskou K. K., Deligianni C., Tsatsanis C. and Eliopoulos A. G. (2014) The gut microbiota in mouse models
of inflammatory bowel disease. Front Cell Infect Microbiol 4: 28

References
Jones-Hall Y. L., Kozik A. and Nakatsu C. (2015) Ablation of tumor necrosis factor is associated withdecreased inflammation and alterations of the microbiota in a mouse model of inflammatory bowel
disease. PLoS One 10: e0119441
Chronic inflammation, characteristic of IBD, is closely related to the prolonged secretion of tumor necrosis
factor (TNF). The effects of TNF in colitis and gut microbiota are not fully characterized and may explain
why anti-TNF therapy is not always successful. Microbial composition was assessed by 16S V3-V4 rRNA
amplicon sequencing in an acute colitis model with wild-type (WT) and tnf-/- mice to elucidate TNF effects incolitis and gut microbiota. Inflammation caused significant differences in microbiota composition according
to mice genotype; absence of TNF resulted in milder colitis and less microbial α-diversity than in WT mice.
The authors conclude that TNF-mediated inflammation modifies gut microbiota and, therefore, combined
therapies that inhibit TNF and alter microbial communities could be beneficial.
Illumina Technology: MiSeq 250 bp PE reads

14

Microbes and metagenomics in human healthMorgan X. C., Kabakchiev B., Waldron L., Tyler A. D., Tickle T. L., et al. (2015) Associations between
host gene expression, the mucosal microbiome, and clinical outcome in the pelvic pouch of patientswith inflammatory bowel disease. Genome Biol 16: 67
Ileal pouch-anal anastomosis (IPAA) surgery for ulcerative colitis (UC) is often complicated by pouchitis
associated with anatomical and microbiota changes that resemble a colon-like environment. How and why
these changes take place, and their relation to UC and IBD, is not fully understood. The authors obtained
paired host microbiomes (by 16S V4 amplicon sequencing) and transcriptomes from a large cohort of IPAA
patients to study the microbiome-host gene expression axis. Microbiomes were variable across individuals
and influenced by clinical variables such as antibiotic therapy; host epithelial transcription was influenced
by tissue location. Associations between microbiome and host transcription patterns were related to thelevel of host tissue inflammation: the strongest microbe-host association pattern was enriched complement
system and cytokine IL-12 pathways inversely correlated with abundance of Bifidobacteria and others.
However, it was not possible to generate a pouchitis outcome model based on microbial composition and/ortranscriptional activity, suggesting that the role of sectional changes in epithelial transcripts may not be critical
for the host-microbiome interface during IPAA.
Illumina Technology: MiSeq v2 175 bp PE reads
Schaubeck M., Clavel T., Calasan J., Lagkouvardos I., Haange S. B., et al. (2015) Dysbiotic gut
microbiota causes transmissible Crohn’s disease-like ileitis independent of failure in antimicrobial
defence.
Gut Intestinal dysbiosis is associated with intestinal inflammatory disease (Crohn’s disease or CD), although
functional explanation of this frequent observation is still missing. Temporal metaproteomic (LC-MS) andmetagenomic (16S rRNA gene) profiles of gut microbiota were obtained from TNFdeltaARE mice, a model that
resembles CD pathology. Disease severity and location were microbiota-dependent: inflammation was absent
in germ-free TNFdeltaARE mice and ileitis (but not colitis) was attenuated after antibiotic treatment. Several
compositional and functional alterations were observed in microbiota communities in inflamed mice, featuresthat were reproducible through microbiome transplantation that resulted in CD-like ileitis accompanied byloss of Paneth cell function. The study provides evidence of a causal role for gut dysbiosis in the developmentof chronic ileal inflammatory disease.
Illumina Technology: MiSeq
Knights D., Silverberg M. S., Weersma R. K., Gevers D., Dijkstra G., et al. (2014) Complex hostgenetics influence the microbiome in inflammatory bowel disease. Genome Med 6: 107
The interplay between host genetics and gut microbiome has been observed in inflammatory diseases,
without distinction of specific disease host alleles in relation to specific microbial taxa. Here, biopsies ofthree IBD cohorts were evaluated through genotyping by Immunochip and 16S rRNA sequencing to identify
relationships in host allele-microbial taxa pairs. The authors showed that the risk allele NOD2 consistently
associated with the bacterial taxa Enterobacteriaceae across the different independent cohorts. They also
identified 48 additional IBD single-nucleotide polymorphisms (SNPs) consistently associated with bacterial
taxa in 2 or 3 of the cohorts. These SNPs are related to genes involved in immune responses (eg, JAK-STAT).
Thus, the pairing of genome-microbiome analysis successfully demonstrates the complex relationships
between altered host functional pathways and microbiome structure in IBD.
Illumina Technology: MiSeq, Infinium Immunochip
Rooks M. G., Veiga P., Wardwell-Scott L. H., Tickle T., Segata N., et al. (2014) Gut microbiome
composition and function in experimental colitis during active disease and treatment-induced
remission. ISME J 8: 1403-1417
IBD patients often receive different individual therapy courses to mitigate disease symptoms and promoteremission. The variability of treatments confuses the effective mechanisms responsible for remission,
because not much is known about how therapy affects gut microbiota. Whole-shotgun metagenomics
and 16S rRNA amplicon sequencing were used to survey the gut microbiome in a mouse model of colitis
during active disease and therapy-induced remission. Microbiome differences between both stages, related
to carbohydrate-energy metabolism, bacterial pathogenesis (motility and signal transduction pathways),and increased capacity for xenobiotic metabolism were reported. Unlike gentamicin and metronidazole,
vancomycin was not useful in promoting symptom amelioration, even though the 3 are often used to treat
C. difficile infection. Immunomodulatory therapy also caused alteration of the microbial community byenrichment of different bacterial taxa. The study identifies specific bacterial taxa and pathways involved withdisease, as well as specific alterations under different therapy regimes in a mouse model of colitis.
Illumina Technology: HiSeq 2000

An overview of recent publications featuring Illumina technology

15

Metabolic Diseases: Diabetes and Obesity
Type-1 (T1D) and Type-2 (T2D) diabetes are multifactorial metabolic disordersthat have been linked to changes in the gut microbiota, which is affected by thesame factors that influence development of the disease in genetically predisposed
individuals.46 Although studies on T1D are not conclusive yet, there is cumulative
evidence of reduced taxa diversity and altered interactions with epithelial receptors
and immune cells. Together, these factors exacerbate autoimmune destruction of46.

Gulden E., Wong F. S. and Wen L. (2015)
The gut microbiota and Type 1 Diabetes. Clin
Immunol 159: 143-153

47.

Hu C., Wong F. S. and Wen L. (2015) Type 1
diabetes and gut microbiota: Friend or foe?
Pharmacol Res 98: 9-15

48.

Labbe A., Ganopolsky J. G., Martoni C. J.,
Prakash S. and Jones M. L. (2014) Bacterial
bile metabolising gene abundance in Crohn’s,
ulcerative colitis and type 2 diabetes metagenomes. PLoS One 9: e115175

49.

Neis E. P., Dejong C. H. and Rensen S.
S. (2015) The role of microbial amino acidmetabolism in host metabolism. Nutrients 7:
2930-2946

50.

Qin J., Li Y., Cai Z., Li S., Zhu J., et al. (2012)
A metagenome-wide association study of gut
microbiota in type 2 diabetes. Nature 490:
55-60

51.

Karlsson F. H., Tremaroli V., Nookaew I.,
Bergstrom G., Behre C. J., et al. (2013) Gut
metagenome in European women with normal,
impaired and diabetic glucose control. Nature
498: 99-103

pancreatic β cells.47
A moderate degree of dysbiosis reduced epithelial permeability and leakage ofinflammatory mediators in T2D, along with reduced abundance of bile metabolizing
genes.48 Excessive production and intake of branched-chain and aromatic aminoacids by an altered microbiota 49 are events associated with the disease. Alteredmetabolic pathways may be useful as diabetes markers,50 due to their contribution todifferential dysbiosis signatures.51

Reviews
Gulden E., Wong F. S. and Wen L. (2015) The gut microbiota and Type 1 Diabetes. Clin Immunol 159: 143-153
Hansen T. H., Gobel R. J., Hansen T. and Pedersen O. (2015) The gut microbiome in cardio-metabolic health.
Genome Med 7: 33
He C., Shan Y. and Song W. (2015) Targeting gut microbiota as a possible therapy for diabetes. Nutr Res 35:
361-367
Hu C., Wong F. S. and Wen L. (2015) Type 1 diabetes and gut microbiota: Friend or foe? Pharmacol Res 98: 9-15
Parekh P. J., Balart L. A. and Johnson D. A. (2015) The Influence of the Gut Microbiome on Obesity,
Metabolic Syndrome and Gastrointestinal Disease. Clin Transl Gastroenterol 6: e91
Gomes A. C., Bueno A. A., de Souza R. G. and Mota J. F. (2014) Gut microbiota, probiotics and diabetes.
Nutr J 13: 60
Nielsen D. S., Krych L., Buschard K., Hansen C. H. and Hansen A. K. (2014) Beyond genetics. Influence of
dietary factors and gut microbiota on type 1 diabetes. FEBS Lett 588: 4234-4243

References
Daft J. G., Ptacek T., Kumar R., Morrow C. and Lorenz R. G. (2015) Cross-fostering immediately after
birth induces a permanent microbiota shift that is shaped by the nursing mother. Microbiome 3: 17
Recognizing the influence of microbiota in metabolic diseases with host genetic components is only a small
aspect of disease complexity. Manipulating the microbiota toward a stable “healthy” phenotype is desirable
and must include gut microbiota establishment. Cross-fostering effects in gut microbiota were surveyed byamplicon sequencing in mouse pups (NOR and NOD strains). Establishment of gut microbiota was quick anddetermined by the nursing mother microbiota, and was stable at 32 weeks. This method can be extended tocurrent models of inflammatory disease and T1D to normalize commensal microbiota among different strainsof mice.
Illumina Technology: MiSeq 250 bp PE reads

16

Microbes and metagenomics in human healthCross-fostering effects in gut microbiota were surveyed by amplicon sequencing in mouse pups.
Bell E. T., Suchodolski J. S., Isaiah A., Fleeman L. M., Cook A. K., et al. (2014) Faecal microbiota ofcats with insulin-treated diabetes mellitus. PLoS One 9: e108729
Cats also suffer from T2D and receive insulin treatment just like humans do. This study evaluated the fecal
microbiota in a small number of insulin-treated diabetic (n = 10) and nondiabetic (n =2 0) cats by 16S V4rRNA sequencing and qPCR. The authors did not observe any differences in gut microbiota composition
or abundance between insulin-treated diabetic and nondiabetic cats. In fact, no differences were observed
according to age, sex, breed, or dietary formulation. Only a decrease in Faecalibacterium spp was identified
by qPCR in cats aged over 10 years. Additional studies are required to investigate differences in larger
cohorts. These studies should compare functional products, transcripts, and metabolic factors that are notreflected by total microbial composition, in diabetic and nondiabetic cats.

52.

Turnbaugh P. J., Ley R. E., Mahowald M.
A., Magrini V., Mardis E. R., et al. (2006) An
obesity-associated gut microbiome withincreased capacity for energy harvest. Nature
444: 1027-1031

53.

Tremaroli V. and Backhed F. (2012) Functional
interactions between the gut microbiota andhost metabolism. Nature 489: 242-249

Illumina Technology: MiSeq

Obesity
Host and environmental factors that influence metabolic diseases highlight therelationship between diet, microbes, and metabolism. For example, obese mice andhumans show reproducible shifts in gut microbial communities. Disturbances in the
microbiota enhance energy harvest by enriching metabolic pathways that yield anexcess of short-chain fatty acids (later deposited in adipose tissue) and energy gain.52
Additionally, products of altered microbial metabolism act as host metabolism signals
in sites outside the intestines, like brain and liver, affecting the level of obesity and its
associated comorbidities.53

Gut microbiome studies in diabetic and obese mouse models reveal complex relationships among diet,inflammation, and metabolism.

An overview of recent publications featuring Illumina technology

17References
Chassaing B., Koren O., Goodrich J. K., Poole A. C., Srinivasan S., et al. (2015) Dietary emulsifiers
impact the mouse gut microbiota promoting colitis and metabolic syndrome. Nature 519: 92-96
Gut microbes are kept at a safe distance from the intestinal epithelium by layers of mucus. Emulsifiers disrupt
this mucus layer in vitro, and their inclusion in processed foods may be a contributing factor for the increased
incidence of metabolic diseases. Gut tissue and microbiomes of mice were exposed to low concentrationsof 2 common emulsifiers, carboxymethylcellulose (CMC) or polysorbate-80 (P80), and monitored by 16S
V4 rRNA sequencing. Both emulsifiers caused mild inflammation and obesity in WT mice, and colitis in amouse-sensitive model. Obesity was correlated with increased microbiota invasion of the epithelial layer,
altered species composition, and increased proinflammatory potential. Fecal transplants into germ-free micecorroborated the role of these altered gut microbiota in the development of metabolic syndrome. The novel
effects of common emulsifiers in the gut environment, and possible association with metabolic syndromes
and inflammatory diseases, need to be reconsidered in light of their increased incidence.
Illumina Technology: MiSeq 250 bp PE reads
Frank D. N., Bales E. S., Monks J., Jackman M. J., MacLean P. S., et al. (2015) Perilipin-2 Modulates
Lipid Absorption and Microbiome Responses in the Mouse Intestine. PLoS One 10: e0131944
Microbiota and metabolic diseases are related, but only a few molecular mechanisms underlying thisrelationship have been unveiled. Among them, cytoplasmic lipid drop protein Perilipin-2 (Plin2) is involved
in lipid metabolism in mice fed with a HFD and may alter gut microbiota during diet changes. Wild-type
and Plin2-null mice were fed a low-fat diet (LFD) or HFD, microbiome composition was monitored by 16S
V2–V3 rRNA sequencing, and physiological parameters were recorded. Plin-2 mice displayed significant
disturbances in physiological parameters when compared to WT controls. Dietary fat content and Plin-2
were independently associated with microbiome composition, diversity, and functional differences. Plin-2
modulates lipid levels in feces and energy utilization in mice, both of which correlate with specific microbiota
communities. The authors conclude that Plin-2 regulates lipid uptake in intestines that leads to gut
microbiome changes associated with diet-induced obesity.
Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq Reagent Kit v3
Yasir M., Angelakis E., Bibi F., Azhar E. I., Bachar D., et al. (2015) Comparison of the gut microbiota ofpeople in France and Saudi Arabia. Nutr Diabetes 5: e153
Dietary habits influence the gut microbiota composition and function, just as an obesogenic microbiota
influences the development of metabolic disease. Fecal microbiota of normal and obese Saudi and European
individuals, who follow very dissimilar diets, were compared through 16S V3–V4 rRNA sequencing. The
differences found between obese and normal cohorts were not conserved between French and Saudi
individuals. The obese French microbiome was less diverse and mostly dominated by Proteobacteria andBacteroidetes when compared to their normal cohort. Obese Saudis were dominated by Firmicutes but were
not different from their normal cohort in terms of diversity. Overall, French individuals had higher diversity
and richness in their microbiome than Saudis. Differences in gut microbiomes from geographically distantpopulations, along with contrast between normal and obese microbiomes, can be informative and add tolarge cohesive efforts to provide integrative views addressing metabolic syndromes.
Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq 250 bp PE reads
Everard A., Lazarevic V., Gaia N., Johansson M., Stahlman M., et al. (2014) Microbiome of prebiotictreated mice reveals novel targets involved in host response during obesity. ISME J 8: 2116-2130
The identification of beneficial microbes and the pathways that prevent or counteract inflammatory
disturbances and metabolic syndromes can improve management of these cases. Additionally, such studies
can support the use of intervention measures, such as prebiotics. Prebiotic treatment was evaluated inmice under normal and HFD through shotgun metagenomics and antimicrobial peptide analysis. HFD andprebiotics significantly altered gut microbiota composition and potential functionality at several phylum levels
relative to normal controls. The authors identified several taxa with differential abundance in both groups, aswell as enriched pathways associated with these taxa. HFD decreased production of Reg3g and PLA2g2 in
the jejunum, whereas prebiotics increased levels of Reg3g and intectin, both related to intestinal epithelium
health and turnover, partially ameliorating the development of metabolic syndrome in mice. This study
shows specific beneficial mechanisms performed by prebiotic-modified gut microbiota, which are interesting
candidates for dietary intervention in metabolic syndromes.
Illumina Technology: TruSeq SBS Kit v3, HiSeq 2000, CASAVA v1.8.2

18

Microbes and metagenomics in human healthWalters W. A., Xu Z. and Knight R. (2014) Meta-analyses of human gut microbes associated withobesity and IBD. FEBS Lett 588: 4223-4233
Several recent publications indicate an intimate relationship between the gut microbiota and metabolic
diseases. The authors surveyed available literature that had employed high-throughput 16S rRNA gene
amplicon sequencing to characterize the gut microbiota in IBD and obesity. IBD showed consistentmicrobiome signatures across different studies, and these microbiomes could distinguish IBD patients fromhealthy individuals. On the contrary, obesogenic microbiota signatures were not consistent across studies,
despite reproducing the disease by fecal transplantation and being statistically significant in each study. This
study highlights the importance and effect of cohort selection and analysis strategies in different clinicalconditions, as they may not have the same microbiota-disease effect size.
Illumina Technology: 16S rRNA gene amplicon sequencing obtained with Illumina technology

Oral Microbiome

54.

Sato Y., Yamagishi J., Yamashita R., ShinozakiN., Ye B., et al. (2015) Inter-Individual Differences in the Oral Bacteriome Are Greater thanIntra-Day Fluctuations in Individuals. PLoS One
10: e0131607

55.

Zheng W., Zhang Z., Liu C., Qiao Y., Zhou
D., et al. (2015) Metagenomic sequencing
reveals altered metabolic pathways in the oral
microbiota of sailors during a long sea voyage.
Sci Rep 5: 9131

56.

Leishman S. J., Do H. L. and Ford P. J. (2010)
Cardiovascular disease and the role of oral
bacteria. J Oral Microbiol 2: 5781

57.

Xu P. and Gunsolley J. (2014) Application ofmetagenomics in understanding oral healthand disease. Virulence 5: 424-432

58.

Zheng H., Xu L., Wang Z., Li L., Zhang J., etal. (2015) Subgingival microbiome in patientswith healthy and ailing dental implants. Sci Rep
5: 10948

59.

Moon J. H., Lee J. H. and Lee J. Y. (2015)
Subgingival microbiome in smokers andnon-smokers in Korean chronic periodontitis
patients. Mol Oral Microbiol 30: 227-241

60.

Shi B., Chang M., Martin J., Mitreva M., Lux
R., et al. (2015) Dynamic changes in thesubgingival microbiome and their potential for
diagnosis and prognosis of periodontitis. MBio
6: e01926-01914

The community of microbes living in the oral cavity performs multiple functions. While
the microbial composition for individuals is relatively stable, it can vary substantiallyamong individuals.54 Altered oral microbiomes are informative of health status, suchas nutritional deficiencies 55 and cardiovascular disease.56
The microbial communities in the oral cavity are organized in compartments andbiofilms. Excessive buildup can form plaque and cause oral diseases. Metagenomic
studies have identified specific metabolic pathways of oral disease.57 During oraldiseases—such as chronic periodontitis, dental caries, and gingivitis—the subgingival
microbiome undergoes dynamic changes that are associated with infection of dental
implants by oral pathogens 58 and smoking.59 These changes can be monitored toassess disease progression and therapy effectiveness.60

During oral diseases—such as chronic periodontitis, dental caries, and gingivitis—the subgingival
microbiome undergoes dynamic changes that can be monitored to assess disease progression andtherapy effectiveness.

Reviews
Duran-Pinedo A. E. and Frias-Lopez J. (2015) Beyond microbial community composition: functional activitiesof the oral microbiome in health and disease. Microbes Infect 17: 505-516
Diaz P. I., Strausbaugh L. D. and Dongari-Bagtzoglou A. (2014) Fungal-bacterial interactions and theirrelevance to oral health: linking the clinic and the bench. Front Cell Infect Microbiol 4: 101
McLean J. S. (2014) Advancements toward a systems level understanding of the human oral microbiome.
Front Cell Infect Microbiol 4: 98

An overview of recent publications featuring Illumina technology

19

Xu P. and Gunsolley J. (2014) Application of metagenomics in understanding oral health and disease.
Virulence 5: 424-432
Zaura E., Nicu E. A., Krom B. P. and Keijser B. J. (2014) Acquiring and maintaining a normal oral microbiome:
current perspective. Front Cell Infect Microbiol 4: 85

References
Edlund A., Yang Y., Yooseph S., Hall A. P., Nguyen D. D., et al. (2015) Meta-omics uncover temporal
regulation of pathways across oral microbiome genera during in vitro sugar metabolism. ISME J
Bacteria-mediated pH drops and subsequent recovery occur upon addition of a carbohydrate source insupragingival plaque. These events are important in dental caries development, but fully characterized
metabolic or transcriptomic details are still missing. The authors developed an in vitro model inoculated withsupragingival plaque microbial communities and analyzed metatranscriptomic and metabolomic profilesof these biofilms. The authors found microbial taxa (Lactobacillus, Streptococcus) and their upregulated
pathways involved in acid neutralization in response to a pH drop. These taxa were also important during
pH recovery of the plaque. Specific metabolites and corresponding transcripts/taxa were identified
along the pH gradient. These signatures, for healthy and disease states, could be used to compare oral
microbiome studies.
Illumina Technology: HiSeq 2000
Sato Y., Yamagishi J., Yamashita R., Shinozaki N., Ye B., et al. (2015) Inter-Individual Differences in theOral Bacteriome Are Greater than Intra-Day Fluctuations in Individuals. PLoS One 10: e0131607
In this study, oral supragingival microbiomes were obtained from healthy individuals at 3 time points
during the day for 3 days and studied by 16S V4 rRNA gene sequencing. The authors showed that oral
microbiomes from healthy individuals were stable throughout the day. In contrast, interindividual variation
was common at the species level. The within-individual stability allowed them to identify 40 co-occurrencesamong rare bacteria.
Illumina Technology: MiSeq Reagent Kit v2 250 bp PE reads
Duran-Pinedo A. E., Chen T., Teles R., Starr J. R., Wang X., et al. (2014) Community-wide
transcriptome of the oral microbiome in subjects with and without periodontitis. ISME J 8: 1659-1672
Periodontitis is a polymicrobial inflammation disease. Metatranscriptomes and metagenomes wereobtained from subgingival samples from healthy and periodontitis-positive individuals to identify community
composition and transcriptome profile differences between both states. The most abundant species withhigher expression of virulence genes found in periodontitis were the known pathogens P. gingivalis, T.
forsythia, and T. denticola. The correlation between abundance and expression was not conserved in othermicroorganisms, and some “healthy” bacteria expressed putative virulence factors. The community displayed
upregulated pathways consistent with periodontitis related to flagellar motility, peptide transport, ironacquisition, β-lactam degradation, and lipid A biosynthesis. These results support the integrated actions of
the community toward disease progression.
Illumina Technology: Nextera XT DNA Library Prep Kit, cBOT, MiSeq v2 150 PE reads, HiSeq 2500

Periodontitis.

20

Microbes and metagenomics in human healthOther Human Biomes
High-throughput technologies and lower costs have enabled research to characterizevarious complex and dynamic human biomes.
The skin is the first surface to come into contact with the environment. Its microbiota

61.

62.

is complex and defined by microenvironments in specific topographical regions of
the body. These microenvironments contribute to particular chemical signatures
63.

influenced by gender 61 and external compounds, such as hygiene products.62
The skin microbiome might be a tool for therapies against cutaneous diseases,
skin cancer, and monitoring health threats or environmental disturbances. The

64.

development of cosmetic and hygiene products may benefit from specific genderand topographic-based signatures that, for example, influence body odor.

65.

66.

67.

68.

69.

The development of cosmetic and hygiene products may benefit from specific gender- and topographicbased signatures that, for example, influence body odor.63

The vaginal microbiome is highly dynamic throughout life and influenced by70.

age, reproductive state, ethnicity, pH, and other factors. Disturbances in taxa
64

abundance and diversity are associated with increased risk of preterm birth,65
gynecologic infections, cancer, and side-effects from chemotherapy and radiation incancer patients.

71.

66

Recently, airway microbiome dynamics have been related to chronic obstructive
pulmonary disease,67 asthma,68 and cystic fibrosis.69, 70, 71, 72, 73, 74
72.

Reviews
Grice E. A. (2014) The skin microbiome: potential for novel diagnostic and therapeutic approaches tocutaneous disease. Semin Cutan Med Surg 33: 98-103

73.

Oh J., Byrd A. L., Deming C., Conlan S., Program N. C. S., et al. (2014) Biogeography and individuality shapefunction in the human skin metagenome. Nature 514: 59-64
Payne M. S. and Bayatibojakhi S. (2014) Exploring preterm birth as a polymicrobial disease: an overview of
the uterine microbiome. Front Immunol 5: 595
Salava A. and Lauerma A. (2014) Role of the skin microbiome in atopic dermatitis. Clin Transl Allergy 4: 33
van de Wijgert J. H., Borgdorff H., Verhelst R., Crucitti T., Francis S., et al. (2014) The vaginal microbiota:
what have we learned after a decade of molecular characterization? PLoS One 9: e105998

74.

Troccaz M., Gaia N., Beccucci S., Schrenzel
J., Cayeux I., et al. (2015) Mapping axillary
microbiota responsible for body odours using aculture-independent approach. Microbiome 3: 3
Bouslimani A., Porto C., Rath C. M., Wang M.,
Guo Y., et al. (2015) Molecular cartography ofthe human skin surface in 3D. Proc Natl Acad
Sci U S A 112: E2120-2129
Oh J., Byrd A. L., Deming C., Conlan S.,
Program N. C. S., et al. (2014) Biogeography
and individuality shape function in the humanskin metagenome. Nature 514: 59-64
Zhou X., Brown C. J., Abdo Z., Davis C. C.,
Hansmann M. A., et al. (2007) Differences in
the composition of vaginal microbial communities found in healthy Caucasian and black
women. ISME J 1: 121-133
Hyman R. W., Fukushima M., Jiang H., Fung
E., Rand L., et al. (2014) Diversity of the vaginal microbiome correlates with preterm birth.
Reprod Sci 21: 32-40
Chase D., Goulder A., Zenhausern F., Monk
B. and Herbst-Kralovetz M. (2015) The
vaginal and gastrointestinal microbiomes ingynecologic cancers: a review of applicationsin etiology, symptoms and treatment. Gynecol
Oncol 138: 190-200
Huang Y. J., Sethi S., Murphy T., Nariya S.,
Boushey H. A., et al. (2014) Airway microbiome dynamics in exacerbations of chronic obstructive pulmonary disease. J Clin Microbiol
52: 2813-2823
Holt P. G. (2015) The mechanism or mechanisms driving atopic asthma initiation: The
infant respiratory microbiome moves to center
stage. J Allergy Clin Immunol 136: 15-22
Harris K. A., Underwood A., Kenna D. T.,
Brooks A., Kavaliunaite E., et al. (2015)
Whole-genome sequencing and epidemiological analysis do not provide evidence forcross-transmission of mycobacterium abscessus in a cohort of pediatric cystic fibrosis
patients. Clin Infect Dis 60: 1007-1016
Hoffman L. R., Pope C. E., Hayden H. S.,
Heltshe S., Levy R., et al. (2014) Escherichia
coli dysbiosis correlates with gastrointestinal
dysfunction in children with cystic fibrosis. Clin
Infect Dis 58: 396-399
Lopez-Collazo E., Jurado T., de Dios CaballeroJ., Perez-Vazquez M., Vindel A., et al. (2015)
In vivo attenuation and genetic evolution of a
ST247-SCCmecI MRSA clone after 13 yearsof pathogenic bronchopulmonary colonization
in a patient with cystic fibrosis: implications of
the innate immune response. Mucosal Immunol 8: 362-371
Turner K. H., Wessel A. K., Palmer G. C.,
Murray J. L. and Whiteley M. (2015) Essential
genome of Pseudomonas aeruginosa in cystic
fibrosis sputum. Proc Natl Acad Sci U S A
112: 4110-4115
Waters V. (2014) Editorial commentary: how
can understanding the phenotype of Pseudomonas aeruginosa lead to more successful
eradication strategies in cystic fibrosis? Clin
Infect Dis 59: 632-634
Whiteson K. L., Meinardi S., Lim Y. W.,
Schmieder R., Maughan H., et al. (2014)
Breath gas metabolites and bacterial metagenomes from cystic fibrosis airways indicate
active pH neutral 2,3-butanedione fermentation. ISME J 8: 1247-1258

An overview of recent publications featuring Illumina technology

21

References
Bouslimani A., Porto C., Rath C. M., Wang M., Guo Y., et al. (2015) Molecular cartography of thehuman skin surface in 3D. Proc Natl Acad Sci U S A 112: E2120-2129
The skin is the largest organ in the human body. Its chemical makeup is likely influenced by skin cells andthe microbes that inhabit it. The authors developed a highly integrated 3D molecular cartography mapof the human body by integrating chemical data with proteomic data (UPLC-QTOF, MALDI-TOF) andmicrobiome composition (16S rRNA sequencing) of the skin according to body site. This 3D map associatedspecific taxa to body site, metabolites, and chemical compounds. It allowed co-occurrence analysis, andrevealed gender-associated characteristics and the impact of hygiene products on human skin. This map
constitutes a detailed panorama of the human skin, and it is potentially useful in research and customized
product development.
Illumina Technology: HiSeq 2000 100 bp PE reads
Boutin S., Graeber S. Y., Weitnauer M., Panitz J., Stahl M., et al. (2015) Comparison of microbiomes
from different niches of upper and lower airways in children and adolescents with cystic fibrosis.
PLoS One 10: e0116029
The relationship between the respiratory tract microbiome and onset/progression of cystic fibrosis (CF) is
not fully understood, despite chronic infections in CF caused by characteristic pathogens. NGS was used tocompare the microbiome of the nasal cavity, throat, and sputum in young CF patients with mild to moderatelung disease. The sputum microbiome between children and adult CF patients was similar, implying that an
adult-like microbiome is established early in life. The high interindividual variation in sputum microbiomes may
be due to an increased abundance of pathogens, such as Pseudomonas aeruginosa. This study shows theeffects of potential pathogens in CF airway microbiota. It also demonstrates that throat swabs may be an
appropriate proxy if sputum sampling is not feasible.
Illumina Technology: HiSeq
Darch S. E., McNally A., Harrison F., Corander J., Barr H. L., et al. (2015) Recombination is a key driverof genomic and phenotypic diversity in a Pseudomonas aeruginosa population during cystic fibrosis
infection. Sci Rep 5: 7649
Pseudomonas aeruginosa causes chronic lung infections in CF patients, with diverging phenotypes inisolates from the same individuals over time. Whole-genome sequencing (WGS) of P. aeruginosa isolates
collected from the same individual at different time points was used to uncover mechanisms responsible forthis phenotypic variation. This study showed intraindividual diversity related to SNPs ranging from 5 to 64,
with evidence of recombination as the main event driving genetic and phenotypic diversity of P. aeruginosa
during in vivo chronic CF infection. The authors discuss these findings and their implications in importantphenotypes, such as antimicrobial resistance patterns.
Illumina Technology: HiSeq 2000 150 bp PE reads

Pseudomonas aeruginosa colonies.
Kramer R., Sauer-Heilborn A., Welte T., Jauregui R., Brettar I., et al. (2015) High individuality of
respiratory bacterial communities in a large cohort of adult cystic fibrosis patients under continuous
antibiotic treatment. PLoS One 10: e0117436
Routine clinical diagnosis of microbial infections in CF patients is restricted to a few known pathogens,despite growing molecular evidence of polymicrobial infections. Amplicon sequencing (16S V3 rRNA gene)
was applied to sputum samples from adults under continuous antibiotic treatment and compared to routine

22

Microbes and metagenomics in human healthculture methods. Communities were diverse and highly individual. Bacteria associated with CF were revealedin finer detail only by NGS, while other methods showed a similar prevalence of known pathogens. There
were no associations between host factors and the CFTR genotype, although non-ΔF508 mutations in the
CFTR gene often showed low abundances of P. aeruginosa. Continuous antibiotic treatment may be thecause of the high individuality and lack of correlation to clinical host factors.

75.

Liu J., Yan R., Zhong Q., Ngo S., Bangayan N.
J., et al. (2015) The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin. ISME J 9: 2078-2093

Illumina Technology: Genome AnalyzerIIx
Liu J., Yan R., Zhong Q., Ngo S., Bangayan N. J., et al. (2015) The diversity and host interactions of
Propionibacterium acnes bacteriophages on human skin. ISME J 9: 2078-2093
Phages are able to regulate microbial populations by inducing lysis, lysogeny, or resistance. This regulation
may also occur in human skin, but relatively little information is available. This study focused on the diversity
and host interactions of the bacteriophages of Propionibacterium acnes. The authors sequenced 48 phages
recovered from acne patients and healthy individuals and analyzed healthy skin metagenomes looking forP. acnes phages. One phage strain at a time dominates skin, although some phages are common among
related and unrelated individuals, which may suggest the existence of a common core of phages in thehuman population and transmission of phages. Furthermore, CRISPR sequences may not be sufficient toconfer resistance against phages in type II P. acnes strains. This study shows how phages can regulate
microbial populations, resulting in individualized microbiomes.
Illumina Technology: MiSeq

Skin with acne. Phages are able to regulate microbial populations by inducing lysis, lysogeny, or resistance.
The authors sequenced 48 phages recovered from acne patients and healthy individuals and analyzed
healthy skin metagenomes looking for P. acnes phages.75
MacIntyre D. A., Chandiramani M., Lee Y. S., Kindinger L., Smith A., et al. (2015) The vaginal
microbiome during pregnancy and the postpartum period in a European population. Sci Rep 5: 8988
Most vaginal microbiome studies have focused on Northern American populations and have often been
associated with specific pregnancy outcomes. The vaginal microbiomes of pregnant British women
were monitored periodically during uncomplicated pregnancy and after partum by 16S V1-V2 rRNA
gene sequencing. Significant changes were observed after partum, characterized by less dominanceof Lactobacillus spp. with increased α-diversity. Some women, mostly Asian and Caucasian, had a L.
jensenii-dominated microbiome; whereas L. gasseri was absent in African women. This study revealed
biogeography and ethnicity as factors in pregnancy and the postpartum vaginal microbiome, contributing toan understanding of the relationships between the microbiome, health, and pregnancy outcomes.
Illumina Technology: MiSeq 300 bp PE reads
Misic A. M., Davis M. F., Tyldsley A. S., Hodkinson B. P., Tolomeo P., et al. (2015) The shared microbiota of
humans and companion animals as evaluated from Staphylococcus carriage sites. Microbiome 3: 2
Staphylococcus species are common commensal residents of the skin in humans and animals. S.
epidermidis can be a reservoir for antimicrobial resistance genes, and methicillin-resistant S. aureus (MRSA)
can cause serious skin and soft-tissue infections (MRSA SSTI). The authors evaluated the skin microbiomeof MRSA SSTI patient households and their companion animals through 16S V4 rRNA gene sequencing.
Habitants in households were more similar to each other in the absence of pets, suggesting that animals
contribute to the household microbial profile. There were no differences in microbiota associated with MRSASSTI, or carriage of other Staphylococcus. The study highlights how the microbiota is shared between petsand humans, adding to diversity and influencing stability over time.
Illumina Technology: MiSeq 150 bp PE reads

An overview of recent publications featuring Illumina technology

23

Silva P. E., Costa P. S., Avila M. P., Suhadolnik M. L., Reis M. P., et al. (2015) Leprous lesion presents
enrichment of opportunistic pathogenic bacteria. Springerplus 4: 187
M. leprae causes leprosy, a chronic disease that affects skin. Changes in skin microbiota composition causedby the disease have not been characterized in the past, but now high-throughput sequencing can address
this question. Skin microbiota of healthy skin (previously published) and leprous lesions were obtained by
16S V3-V4 rRNA gene and Sanger sequencing. Profound shifts in skin microbiota taxa composition andabundance were associated with leprous lesions. These shifts favored overgrowth of potentially pathogenic
bacteria not usually associated with normal skin (Burkhordelia, Pseudomonas and Bacillus) at the expense of
normal flora (Propionibacterium, Staphylococcus and Corynebacterium). The results suggest that potentiallypathogenic bacteria may have gained a competitive advantage over normal resident microbes in theenvironment provided by leprous lesions.
Illumina Technology: MiSeq
Ghartey J. P., Smith B. C., Chen Z., Buckley N., Lo Y., et al. (2014) Lactobacillus crispatus dominantvaginal microbiome is associated with inhibitory activity of female genital tract secretions against
Escherichia coli. PLoS One 9: e96659
Female genital tract secretions can inhibit growth or prevent colonization of potential pathogens. These
cooperative activities can yield biomarkers of a healthy microbiome. Vaginal secretions from nonpregnant andnear-term pregnant women were sampled and characterized by microbiome sequencing (16S V6 amplicon)
and inhibitory activity against E. coli. There was no overall difference in microbiome composition between
pregnant and nonpregnant women. E. coli inhibitory secretions were enriched in L. crispatus, and culture
supernatants reproduced this effect in vitro with variation among different L. crispatus strains. This study
shows the protective effects against E. coli colonization in microbiomes rich in L. crispatus, suggesting that
promoting these environments may prevent negative pregnancy outcomes.
Illumina Technology: HiSeq 2000 100 bp PE reads
Walther-Antonio M. R., Jeraldo P., Berg Miller M. E., Yeoman C. J., Nelson K. E., et al. (2014)
Pregnancy’s stronghold on the vaginal microbiome. PLoS One 9: e98514
Influence of the vaginal microbiome in pregnancy outcome has been suggested by previous studies withsome limitations as to length of experimental observation. In this study, the vaginal microbiomes of womencarrying uncomplicated pregnancies were monitored at intervals throughout the complete pregnancy
period by 16S V3-V5 rRNA sequencing. Vaginal microbiomes were stable, exhibited low diversity, and were
dominated by Lactobacillus genus, specifically L. crispatus, in both Caucasian and African-American women.
Very few samples were dominated or shifted towards L. liners. However, some observed differences were
attributed to ethnicity. Caucasian microbiomes clustered by trimester and progressed toward a common
attractor, whereas African-American microbiomes clustered by individual. The study supports previous
observations about vaginal microbiome characteristics and speculates about the influence of ethnicity on
microbiome differences contributing to the risk for complications of pregnancy.
Illumina Technology: MiSeq 250 bp PE reads

Vaginal microbiomes are dominated by the genus Lactobacillus. Disturbances influence pregnancy
outcomes and may contribute to the risk for complications of pregnancy.

24

Microbes and metagenomics in human health

VIROMES AND HUMAN HEALTH
Viral Populations
Direct sequencing, also called shotgun metagenomics, is used to identify viruses
and determine their abundance in microbial communities.76 It has the distinctive

76.

Allen H. K., Bunge J., Foster J. A., Bayles D.
O. and Stanton T. B. (2013) Estimation of viralrichness from shotgun metagenomes using afrequency count approach. Microbiome 1: 5

77.

Sridhar S., To K. K., Chan J. F., Lau S. K.,
Woo P. C., et al. (2015) A systematic approach
to novel virus discovery in emerging infectious
disease outbreaks. J Mol Diagn 17: 230-241

78.

Rosseel T., Ozhelvaci O., Freimanis G. and
Van Borm S. (2015) Evaluation of convenient
pretreatment protocols for RNA virus metagenomics in serum and tissue samples. J Virol
Methods 222: 72-80

79.

Hall R. J., Wang J., Todd A. K., Bissielo A.
B., Yen S., et al. (2014) Evaluation of rapid
and simple techniques for the enrichment ofviruses prior to metagenomic virus discovery. J
Virol Methods 195: 194-204

80.

Stremlau M. H., Andersen K. G., Folarin O. A.,
Grove J. N., Odia I., et al. (2015) Discovery ofnovel rhabdoviruses in the blood of healthy
individuals from West Africa. PLoS Negl Trop
Dis 9: e0003631

81.

Qin X. C., Shi M., Tian J. H., Lin X. D., Gao D.
Y., et al. (2014) A tick-borne segmented RNA
virus contains genome segments derived from
unsegmented viral ancestors. Proc Natl Acad
Sci U S A 111: 6744-6749

82.

Walker P. J., Firth C., Widen S. G., Blasdell
K. R., Guzman H., et al. (2015) Evolution of
genome size and complexity in the rhabdoviridae. PLoS Pathog 11: e1004664

83.

Colson P., La Scola B. and Raoult D. (2013)
Giant viruses of amoebae as potential humanpathogens. Intervirology 56: 376-385

84.

Kernbauer E., Ding Y. and Cadwell K. (2014)
An enteric virus can replace the beneficial
function of commensal bacteria. Natureadvance online publication:

85.

Abeles S. R., Robles-Sikisaka R., Ly M., Lum
A. G., Salzman J., et al. (2014) Human oral viruses are personal, persistent and gender-consistent. ISME J 8: 1753-1767

advantages of being able to detect novel viruses and avoid viral mutations during
cell culture adaptation, a common concern for standard isolation and propagation
techniques.77 Viruses do not have a phylogenetic marker analogous to the bacterial
16S rRNA gene, so targeted sequencing based on the 16S rRNA gene will notdetect viruses.
In clinical samples, various protocols are used to increase nucleic acid yield for
discovery and detection of viruses.78 Enrichment and cDNA synthesis are particularly
important to detect RNA viruses

79, 80such as Ebola virus (EBOV), severe acuterespiratory syndrome coronavirus (SARS-CoV), Middle East respiratory coronavirus(MERS-CoV), rabies, and many others that dwell in the environment within vector
hosts.81, 82 Analysis of metagenomic data from environmental and human sourcesshows the presence of giant viruses of amoebae, which may be linked to pneumonia
of unknown etiology in humans.83
In humans, eukaryotic and prokaryotic viruses shape the intestinal microbiota,84
and a human virome is recognized as characteristically persistent and particular toeach individual.85

Reviews
Sridhar S., To K. K., Chan J. F., Lau S. K., Woo P. C., et al. (2015) A systematic approach to novel virus
discovery in emerging infectious disease outbreaks. J Mol Diagn 17: 230-241
McElroy K., Thomas T. and Luciani F. (2014) Deep sequencing of evolving pathogen populations: applications,errors, and bioinformatic solutions. Microb Inform Exp 4: 1
Quinones-Mateu M. E., Avila S., Reyes-Teran G. and Martinez M. A. (2014) Deep sequencing: becoming a
critical tool in clinical virology. J Clin Virol 61: 9-19
Virgin H. W. (2014) The virome in mammalian physiology and disease. Cell 157: 142-150

References
Rosseel T., Ozhelvaci O., Freimanis G. and Van Borm S. (2015) Evaluation of convenient pretreatment
protocols for RNA virus metagenomics in serum and tissue samples. J Virol Methods 222: 72-80
A thorough evaluation of pretreatment methods is needed to provide uniform guidelines for RNA virus
metagenomics. In this study, an RNA virus was spiked, in low and high concentrations, in serum and lungtissue samples to evaluate common pretreatment and enrichment methods for viral sequences. The authors
found that virion enrichment strategies (filtering and DNase treatment) are beneficial. In serum samples,
DNase treatment coupled to rRNA depletion provided the best gain in viral sequences. In tissue samples,rRNA depletion obtained the biggest gain. The authors highlight that different samples benefit from different
enrichment protocols to obtain optimal nucleic acid concentration and quality for metagenomic analysis.
Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq v3 300 bp PE reads

An overview of recent publications featuring Illumina technology

25

Xu G. J., Kula T., Xu Q., Li M. Z., Vernon S. D., et al. (2015) Viral immunology. Comprehensive
serological profiling of human populations using a synthetic human virome. Science 348: aaa0698
Serological tests are performed based on clinical presumption about the identity of a single (or small numberof) infectious agent(s). Assumption-free tests that can determine serological responses to current and past
pathogens can be useful in clinical settings and epidemiology studies at the population level. The authors
have developed Systematic Viral Epitope Scanning or VirScan, a high-throughput method to analyze theinventory of antiviral antibodies using single drop of blood. The method relies on immunoprecipitation andmassively parallel sequencing of phage libraries displaying viral peptides from all currently known humanviruses, representing 206 human viral species. Tested in 569 individuals from around the world, VirScan
identified antibodies to an average of 10 viruses per person and 84 viral species in 2 individuals. Some
peptides were consistently targeted by antibodies and may represent “public epitopes.”
Illumina Technology: HiSeq 2000 50 bp PE reads
Bzhalava D., Muhr L. S., Lagheden C., Ekstrom J., Forslund O., et al. (2014) Deep sequencing extends
the diversity of human papillomaviruses in human skin. Sci Rep 4: 5807
Human papillomaviruses (HPVs) are abundant in and on skin. Previous metagenomic analyses have shown
273 cutaneous HPVs, including 47 previously unknown types. The authors used Illumina sequencing toobtain deeper sequencing, without prior PCR amplification, in pools of skin lesions. They identified 23 known
HPV types, 3 novel putative HPVs, and 4 non-HPV viruses. They detected 385 HPV types, including 226
previously unknown types, in 326 PCR-amplified samples of skin lesions. The authors conclude that the
number of HPV types will likely increase as deep sequencing technologies improve in performance and cost.
Illumina Technology: TruSeq Nano DNA Library Prep Kit, Nextera DNA Library Prep Kit, MiSeq v2 250 bp
PE reads, MiSeq v3 300 bp PE reads

Viral Zoonotic Reservoirs
Zoonotic reservoirs play a significant role in the dissemination of pathogens. With
NGS, it is now possible to screen reservoir animals to predict and prevent viralpathogen outbreaks.
Bat viromes are highly variable within and among species, but colonies maintainviral species in the wild.86 The bat virome also contains vertebrate pathogens suchas influenza A, hepaciviruses, hepadnaviruses, hantaviruses, cedar virus, SARS-likebetacoronaviruses,87 papillomavirus, picornavirus, and polyomavirus.88 Bats areimportant reservoirs for highly pathogenic emerging viruses (Table 1).

Bat stew.

26

Microbes and metagenomics in human health86.

Dacheux L., Cervantes-Gonzalez M., Guigon
G., Thiberge J. M., Vandenbogaert M., et al.
(2014) A preliminary study of viral metagenomics of French bat species in contact withhumans: identification of new mammalianviruses. PLoS One 9: e87194

87.

Quinones-Mateu M. E., Avila S., Reyes-Teran
G. and Martinez M. A. (2014) Deep sequencing: becoming a critical tool in clinical virology.
J Clin Virol 61: 9-19

88.

Wang J., Moore N. E., Murray Z. L., McInnes
K., White D. J., et al. (2015) Discovery of novel
virus sequences in an isolated and threatened
bat species, the New Zealand lesser shorttailed bat (Mystacina tuberculata). J Gen Virol
96: 2442-2452

Table 1: Pathogenic Viruses in Bats

Common Name

Scientific Name

Virus

Reference

Chinese horsehoe bats

Genus Rhinolophus

SARS-CoV89

Egyptian tomb bat

Taphozous perforatus

MERS-CoV

90

Fruit bats

Hypsignathus monstrosus, Epomops
franqueti, and Myonycteris torquata

EBOV

91, 92Arthropods play important roles in ecology and transmission of infectious diseases.93
They harbor a diversity of pathogens that affect mammals, along with nonpathogen

89.

Ge X. Y., Li J. L., Yang X. L., Chmura A. A.,
Zhu G., et al. (2013) Isolation and characterization of a bat SARS-like coronavirus that uses
the ACE2 receptor. Nature 503: 535-538

90.

Milne-Price S., Miazgowicz K. L. and Munster
V. J. (2014) The emergence of the Middle East
respiratory syndrome coronavirus. Pathog Dis
71: 121-136

91.

Leroy E. M., Epelboin A., Mondonge V.,
Pourrut X., Gonzalez J. P., et al. (2009) Human
Ebola outbreak resulting from direct exposure
to fruit bats in Luebo, Democratic Republic ofCongo, 2007. Vector Borne Zoonotic Dis 9:723-728

92.

Leroy E. M., Kumulungui B., Pourrut X., Rouquet P., Hassanin A., et al. (2005) Fruit bats as
reservoirs of Ebola virus. Nature 438: 575-576

93.

Bichaud L., de Lamballerie X., Alkan C., Izri
A., Gould E. A., et al. (2014) Arthropods as asource of new RNA viruses. Microb Pathog
77: 136-141

94.

Ponnusamy L., Gonzalez A., Van Treuren W.,
Weiss S., Parobek C. M., et al. (2014) Diversityof Rickettsiales in the microbiome of the lonestar tick, Amblyomma americanum. Appl
Environ Microbiol 80: 354-359

95.

McBride C. S., Baier F., Omondi A. B., Spitzer
S. A., Lutomiah J., et al. (2014) Evolution ofmosquito preference for humans linked to an
odorant receptor. Nature 515: 222-227

symbionts in primitive gut compartments.94 Unlike bats, some mosquitoes are actively
attracted to humans and may have evolved this preference based on expressionof human-specific odorant receptors. In turn, human body odor is influenced byskin microbiota. This is an extraordinary example of behavioral specialization.95
Understanding the biology of these interactions is critical when designing rational
measures to control infectious diseases transmitted by vectors. This approach cancertainly be invaluable for molecular epidemiology studies that track changes in viralcommunities in wildlife populations to identify and predict outbreaks.

Reviews
Han H. J., Wen H. L., Zhou C. M., Chen F. F., Luo L. M., et al. (2015) Bats as reservoirs of severe emerging
infectious diseases. Virus Res 205: 1-6
Huang J. H., Jing X. and Douglas A. E. (2015) The multi-tasking gut epithelium of insects. Insect Biochem
Mol Biol
Liang G., Gao X. and Gould E. A. (2015) Factors responsible for the emergence of arboviruses; strategies,
challenges and limitations for their control. Emerg Microbes Infect 4: e18
Narasimhan S. and Fikrig E. (2015) Tick microbiome: the force within. Trends Parasitol 31: 315-323
Bichaud L., de Lamballerie X., Alkan C., Izri A., Gould E. A., et al. (2014) Arthropods as a source of new RNA
viruses. Microb Pathog 77: 136-141
O’Shea T. J., Cryan P. M., Cunningham A. A., Fooks A. R., Hayman D. T., et al. (2014) Bat flight and zoonotic
viruses. Emerg Infect Dis 20: 741-745

References
Chandler J. A., Thongsripong P., Green A., Kittayapong P., Wilcox B. A., et al. (2014) Metagenomic
shotgun sequencing of a Bunyavirus in wild-caught Aedes aegypti from Thailand informs theevolutionary and genomic history of the Phleboviruses. Virology 464-465: 312-319
The Aedes aegypti mosquito, a vector for many viral diseases in human health, remains apparently unaffectedby the many viruses it carries. RNA metagenomic shotgun sequencing coupled to de novo assembly of wild
A. aegypti samples yielded the nearly complete genome of a novel member of the Phlebovirus genus. Thisparticular virus lacks a gene necessary for virulence in vertebrates that may restrict it exclusively to arthropod
hosts. This study sheds light on the evolution of viral host tropicity, as well as the utility of deep metagenomic
sequencing for viral identification.
Illumina Technology: TruSeq Stranded RNA Library Prep Kit, Genome AnalyzerIIx 50 bp single-end (SE) reads

An overview of recent publications featuring Illumina technology

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Dacheux L., Cervantes-Gonzalez M., Guigon G., Thiberge J. M., Vandenbogaert M., et al. (2014) A
preliminary study of viral metagenomics of French bat species in contact with humans: identificationof new mammalian viruses. PLoS One 9: e87194
Studying the bat virome might be the key to preventing outbreaks of viral diseases transmitted to humans
by these vectors. In this study, tissue samples (brain, liver, and lung) were collected from a small cohortof carcasses of insectivorous bats that were found dead or died shortly after collection as part of a rabies
surveillance program. Samples were subjected to high-throughput sequencing to obtain the total viromes.
Among the viruses found were several known viruses able to infect mammals (Retroviridae, Herpesviridae,
and Flaviviridae). New mammalian viruses were also identified, including rotaviruses, bornaviruses,
bunyaviruses, and a novel bat nairovirus. The high diversity and abundance of known and novel mammalianviruses in such a small sample cohort, which was in contact with humans, demonstrate the importance ofbats as viral reservoirs.
Illumina Technology: TruSeq DNA Library Prep Kit v2, TruSeq PE Cluster Kit v3, TruSeq SBS Kit v3,
HiSeq 2000 100 bp PE reads
He B., Zhang Y., Xu L., Yang W., Yang F., et al. (2014) Identification of diverse alphacoronaviruses andgenomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in
China. J Virol 88: 7070-7082
Several severe acute respiratory syndrome-like coronaviruses (SARS-like CoVs) have been identified in bats
previously, but none of their receptor-binding domains are similar to human SARS-CoV, implying that they are
unlikely progenitors of this virus. Here, the authors performed metagenomic analysis of 268 bat rectal swabs
in China and identified α- and β-coronavirus sequences. Full genomic analysis of a SARS-like CoV (LYRa11)
revealed 91% nucleotide identity with human SARS-CoV. The highest identity was found in the S gene,
especially in the receptor-binding domain region. SARS-convalescent human sera recognized the S1 domainof this virus, showing antigenic similarity. The authors suggest that LYRa11 is likely a recombinant descended
from parental lineages that evolved into a number of bat SARS-like CoVs.
Illumina Technology: Genome Analyzer
McBride C. S., Baier F., Omondi A. B., Spitzer S. A., Lutomiah J., et al. (2014) Evolution of mosquito
preference for humans linked to an odorant receptor. Nature 515: 222-227
Aedes aegypti mosquitoes carry several pathogenic viruses that infect humans. For unknown reasons, someof these mosquitoes prefer humans (“domestic form”) instead of nonhuman animals (“forest form”). These 2
mosquito forms were raised in laboratory colonies. RNA-Seq of antennae tissue, the major olfactory organ,
showed that preference for humans is linked to increased expression and sensitivity of the odorant receptor
AaegOr4, which recognizes the abundant human odorant sulcatone. This sensitivity to a human odor compoundis an evolutionary adaptation that has enabled mosquitoes to detect and prefer human hosts. However, neither
odorant receptor nor sulcatone alone are likely to be the only factors that contribute to this behavior.
Illumina Technology: TruSeq RNA Library Prep Kit v2, Illumina GEX and HiSeq 2000

The preference of some mosquitos for humans is linked to increased expression and sensitivity of theodorant receptor AaegOr4, which recognizes the abundant human odorant sulcatone.

28

Microbes and metagenomics in human healthWoo P. C., Lau S. K., Teng J. L., Tsang A. K., Joseph M., et al. (2014) Metagenomic analysis of viromesof dromedary camel fecal samples reveals large number and high diversity of circoviruses andpicobirnaviruses. Virology 471-473: 117-125
The emergence of MERS-CoV, and the presence of neutralizing antibodies against this virus in dromedaries,
prompts questions about the role of these animals in spread of infectious viral diseases. This study focusedon the fecal virome obtained by metagenomic sequencing of 203 dromedaries in Dubai. The authors
identified a wide array of mammalian viruses (Picobirnaviridae, Circoviridae, Picornaviridae, Parvoviridae,
Astroviridae, and Herpesviridae), some of which had not been described previously in dromedaries, andgenerated 14 complete Circoviridae genomes. The large diversity of viruses in dromedary feces demands
further studies to establish their incidence and effects on dromedary and human health.
Illumina Technology: Nextera XT DNA Library Prep Kit, Nextera XT Index Kit, HiSeq 2500 150 bp PE reads
Yang L., Wu Z., Ren X., Yang F., Zhang J., et al. (2014) MERS-related betacoronavirus in Vespertilio
superans bats, China. Emerg Infect Dis 20: 1260-1262
To control transmission of MERS-CoV to humans, it is necessary to find natural viral reservoirs. The authors
collected 32 anal swab samples from Vespertilio superans bats in China that were pooled and processedby virus particle–protected nucleic acid purification and NGS. They identified and generated a draft genomeof a novel betacoronavirus lineage C that shares 60-97% amino acid identity with MERS-CoV, being closely
related to human and camel MERS-CoV. PCR amplicon sequencing showed that 5 samples were positive forthe virus; these samples shared over 98% nucleotide identity with each other. This new lineage is the closest
resembling MERS-CoV at the time of the study, adding to other investigations that place bats as the natural
reservoirs of MERS-CoV.
Illumina Technology: Genome AnalyzerIIx
Zhuang L., Zhang Z., An X., Fan H., Ma M., et al. (2014) An efficient strategy of screening forpathogens in wild-caught ticks and mosquitoes by reusing small RNA deep sequencing data.
PLoS One 9: e90831
Small RNAs may have regulatory roles in viral-host interactions. The authors set out to reanalyze small RNA–
derived sequencing data, obtained from ticks and mosquitoes in Beijing, to screen for viral, prokaryotic, andeukaryotic pathogens. A novel bioinformatic pipeline was developed in house to search for putative pathogen
sequences within the data, and nested PCR was used to confirm some of the results. This method identified
a novel Rickettsia spp in H. longicornis ticks, as well several other putative prokaryotic and eukaryotic
pathogens of interest. Reanalysis of sRNA deep sequencing data could be potentially useful to discover novel
agents or track the origin of pathogens.
Illumina Technology: Genome AnalyzerIIx
Wu Z., Yang L., Ren X., He G., Zhang J., et al. (2015) Deciphering the bat virome catalog to better
understand the ecological diversity of bat viruses and the bat origin of emerging infectious
diseases. ISME J
Identifying the catalog of viruses that reside in bats might be useful to understand emerging infectious
diseases in humans. Here, complete viromes were obtained by high-throughput sequencing of pharyngeal
and anal swabs from 4440 bats of 40 major bat species throughout China. The authors show, in the context
of bat ecological and biological diversity, an in-depth genetic survey that reveals novel bat viruses as well
as viruses closely related to human or animal pathogens. This survey also provides a basis for the origin or
evolution of certain viruses and ecological information for predicting and tracing emerging pathogens.
Illumina Technology: Genome AnalyzerIIx

The virome of bats, like the fruit bats depicted above, harbors a wide array of viruses able to infect humans.

An overview of recent publications featuring Illumina technology

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DNA Viruses
High-throughput sequencing of environmental and clinical samples reveals a diverse
virosphere, where DNA viruses present a broad host range, from hyperthermophilicarchaea to mammal vertebrates.96 NGS is an important tool for discovering novel
viruses, new genes, and associated functions and niches.

References
Jensen R. H., Mollerup S., Mourier T., Hansen T. A., Fridholm H., et al. (2015) Target-dependentenrichment of virions determines the reduction of high-throughput sequencing in virus discovery.
PLoS One 10: e0122636
High-throughput sequencing usually requires virion concentration and enrichment for viral sequence recovery.
The sensitivity and variability of these approaches have not been fully assessed and may be variable. This
study evaluated different library preparation techniques, targeting both RNA and DNA, with and without virion
enrichment. Recovery of DNA and RNA viral sequences benefited from virion enrichment, especially for DNA
viruses, resulting in reduced sequencing efforts. Despite this finding, it was not evident that a lower detectionlevel was achieved when compared to direct metagenomic sequencing.
Illumina Technology: Nextera XT DNA Library Prep Kit, ScriptSeq v2 RNA-Seq Library Prep Kit, HiSeq
2000 100 bp PE reads
Krupovic M., Zhi N., Li J., Hu G., Koonin E. V., et al. (2015) Multiple layers of chimerism in a singlestranded DNA virus discovered by deep sequencing. Genome Biol Evol 7: 993-1001
Single stranded DNA (ssDNA) viruses can infect many organisms, are highly abundant and diverse, cantolerate high nucleotide substitution rates, and perform recombination. A group of ssDNA viruses appears
genomically chimeric because it combines capsid and replicative genes inherited from RNA and other ssDNA
viruses. A new layer of chimerism was exposed in this study by NGS of spin-column–associated CHIV14
ssDNA virus. The replicative genes of this putative new virus are chimeras of functional domains inheritedfrom viruses of different families (RNA and DNA). These chimeric genes may be the product of horizontal
gene transfer and domain shuffling, giving rise to this unusual type of chimeric ssDNA virus.
Illumina Technology: HiSeq 2000
Markus A., Lebenthal-Loinger I., Yang I. H., Kinchington P. R. and Goldstein R. S. (2015) An in vitro
model of latency and reactivation of varicella zoster virus in human stem cell-derived neurons. PLoS
Pathog 11: e1004885
Varicella zoster virus latent infection in ganglia can cause herpes zoster upon reactivation. However, there are
no in vitro models to study this latency-reactivation mechanism. The authors present an in vitro model basedon human neurons derived from embryonic stem cells that undergo stable acyclovir-induced latency, followedby reactivation upon withdrawal of growth factors or inhibition of phosphoinositide-3 kinase activity. RNA-Seq
was used to monitor gene expression during latency and reactivation, showing preferential transcription of
specific genome regions. Interestingly, reducing temperature to 34°C resulted in enhanced viral activation.
This system could prove useful for modeling latency-activation and as a tool to assess therapeutic options.
Illumina Technology: TruSeq Stranded Total RNA LT Library Prep Kit, HiSeq 2500
Chen J., Xue Y., Poidinger M., Lim T., Chew S. H., et al. (2014) Mapping of HPV transcripts in fourhuman cervical lesions using RNAseq suggests quantitative rearrangements during carcinogenic
progression. Virology 462-463: 14-24
Human papillomavirus (HPV) 16 is associated with high risk of cervical cancer, while HPV6b is associatedwith benign lesions in the anogenital tract. However, the drivers for these phenotypic differences are notfully understood. This study used RNA-Seq to generate complete transcriptomes of both viruses in clinicallesions. Both viral types, in their associated lesions, showed intrinsic transcriptomic differences, particularly inpromoter usage. In case of HPV16, defined transcriptomic signatures characterized progression of disease
that were mostly related to regulatory proteins, such as E7, E2, E1, and E5. These specific signatures could
potentially be used as biomarkers for disease progression and prognosis.
Illumina Technology: TruSeq RNA Library Prep Kit v2, HiSeq 2000 v3 50 bp PE reads

30

Microbes and metagenomics in human health96.

DiMaio F., Yu X., Rensen E., Krupovic M.,
Prangishvili D., et al. (2015) Virology. A virusthat infects a hyperthermophile encapsidates
A-form DNA. Science 348: 914-917

RNA Viruses
This group of viruses is characterized by a constrained genome size as well as
host, vector, and genomic diversity. RNA viruses also have a striking ability to97.

Hoffmann B., Tappe D., Hoper D., Herden C.,
Boldt A., et al. (2015) A Variegated Squirrel
Bornavirus Associated with Fatal Human
Encephalitis. N Engl J Med 373: 154-162

98.

Belshaw R., Pereira V., Katzourakis A., Talbot
G., Paces J., et al. (2004) Long-term reinfection of the human genome by endogenous
retroviruses. Proc Natl Acad Sci U S A 101:4894-4899

99.

Grow E. J., Flynn R. A., Chavez S. L., Bayless
N. L., Wossidlo M., et al. (2015) Intrinsic
retroviral reactivation in human preimplantationembryos and pluripotent cells. Nature 522:
221-225

undergo rapid evolution by recombination, reassortment, and conservative aminoacid substitution in changing environments. Many important viral infections are
due to RNA viruses, and NGS is becoming an important tool for viral discovery
and epidemiology. This is especially true for unknown illnesses from unidentified
microorganisms or unsuspected sources. For example, a metagenomic approach
incorporating NGS showed recent transmission of Bornavirus VSBV-1 fromvariegated squirrels to human hosts who had developed fatal encephalitis.97

A metagenomic approach incorporating NGS showed recent transmission of Bornavirus VSBV-1 fromvariegated squirrels to human hosts who had developed fatal encephalitis.

Some ancient RNA viruses have positive effects in human health and development.
Endogenous retroviruses constitute about 8% of the human genome.98 They aremostly silenced, except during certain cancers, human immunodeficiency virus(HIV) infections, and during early embryogenesis when expression may protect thedeveloping embryo from viral infections.99

References
Andersen K. G., Shapiro B. J., Matranga C. B., Sealfon R., Lin A. E., et al. (2015) Clinical Sequencing
Uncovers Origins and Evolution of Lassa Virus. Cell 162: 738-750
The authors generated a genomic catalog of almost 200 Lassa virus (LASV) sequences from clinical androdent reservoir samples. LASV infections mainly result from reservoir-to-human infections. They followed the
spread of LASV across West Africa and showed that this migration was accompanied by changes in LASV
genome abundance, fatality rates, codon adaptation, and translational efficiency.
Illumina Technology: HiSeq 2000 and Nextera XT libraries
Brown J. R., Morfopoulou S., Hubb J., Emmett W. A., Ip W., et al. (2015) Astrovirus VA1/HMO-C: an
increasingly recognized neurotropic pathogen in immunocompromised patients. Clin Infect Dis 60:
881-888
When conventional methods fail to identify the cause, encephalopathy of unknown etiology is a great concern
in clinical settings. Fast and reliable tools are needed to address these cases. Brain biopsy obtained froman immunosuppressed child with encephalopathy of unknown origin was subjected to RNA-Seq, identifying
VA1/HMO-C astrovirus. The finding was confirmed as the cause of encephalopathy by immunohistochemistry,
and the virus was also found in other bodily fluids. These results prompted a survey of 680 stool and 349
cerebrospinal fluid samples that identified a similar virus in another immunosuppressed child. The authors
conclude that this type of astrovirus is neuropathic and that RNA-Seq is a valuable diagnostic tool in
unexplained encephalitis cases.
Illumina Technology: MiSeq

An overview of recent publications featuring Illumina technology

31

Grow E. J., Flynn R. A., Chavez S. L., Bayless N. L., Wossidlo M., et al. (2015) Intrinsic retroviralreactivation in human preimplantation embryos and pluripotent cells. Nature 522: 221-225
Human endogenous retrovirus type K (HERV-K) is the most recently acquired endogenous retrovirus thathas retained multiple copies of complete open reading frames in human cells. The virus is mostly silenced
in cells, except under specific conditions such as HIV-1 infection. This study used RNA-Seq and individualnucleotide resolution cross-linking and immunoprecipitation (iCLIP) analysis to show that hypomethylation oflong terminal repeats and transactivation by OCT4 induce expression of HERV-K transcripts in early embryos.
Expression of some HERV-K products (e.g., Rec) seems to inhibit viral infection in pluripotent cells throughexpression of interferon-induced transmembrane protein 1 (IFITM1) on the cell surface and it also regulates a
set of cellular RNAs. These results indicate that HERV-K may have a protective antiviral role in early embryo
development and interact with host cell factors to regulate embryogenesis.
Illumina Technology: HiSeq 2000, HiSeq 2500
Stenglein M. D., Jacobson E. R., Chang L. W., Sanders C., Hawkins M. G., et al. (2015) Widespreadrecombination, reassortment, and transmission of unbalanced compound viral genotypes in naturalarenavirus infections. PLoS Pathog 11: e1004900
Arenaviruses are segmented RNA viruses (L and S segments) that infect snakes—and, occasionally, rodents
and humans—causing hemorrhagic fever. Events like mutation, recombination, and reassortment occur insegmented RNA viruses and may be the origin of the emergence of mammalian arenaviruses. These events
have not been determined in snakes. Metagenomic sequencing was used to determine arenavirus diversity in
48 naturally infected captive snakes. A total of 23 L and 11 S genotypes were found, and snakes commonly
had multiple infections. The S/L ratio was always imbalanced, with L segment genotypes outnumbering the
S genotypes. Genomic evidence of recombination and reassortment events were common and even atypicalstructures, able to replicate and transmit, were observed. The authors speculate that human intervention, bycommingling snakes previously infected with these viruses, has resulted in this great viral diversity.
Illumina Technology: HiSeq 2500 135 bp PE reads

Arenaviruses are segmented RNA viruses that infect snakes and, occasionally, rodents and humans.
Stremlau M. H., Andersen K. G., Folarin O. A., Grove J. N., Odia I., et al. (2015) Discovery of novel
rhabdoviruses in the blood of healthy individuals from West Africa. PLoS Negl Trop Dis 9: e0003631
The authors used NGS to discover RNA viruses in blood samples of healthy (n = 328) and unexplained febrile
individuals (n = 195) in Nigeria. Febrile individuals carried sequences from a variety of viruses (eg, HIV-1,
Lassa virus). More interestingly, 2 healthy individuals carried 2 novel rhabdoviruses (Ekpoma virus [EKV]-1 andEKV-2) that were highly divergent from other known rhabdoviruses and each other. The closest known virus
is the Bas-Congo virus. Serological surveys in healthy individuals showed that seroprevalence of EKV-2 washigher than that of EKV-1, although cross-reactivity against other rhabdoviruses could not be ruled out.
Illumina Technology: HiSeq 2500 100 bp PE reads
Bhat R. K., Rudnick W., Antony J. M., Maingat F., Ellestad K. K., et al. (2014) Human endogenous
retrovirus-K(II) envelope induction protects neurons during HIV/AIDS. PLoS One 9: e97984
HERV-K is mostly silenced, except under specific conditions. This study aimed to elucidate the role of
HERV-K Env expression in the brain during HIV-1 infection. RNA sequences encoded by HERV-K wereamong the most abundant in brain, particularly in cultured human neurons and in HIV-1–infected brains.
Expression of Env in neuronal cells increased cellular viability and prevented neurotoxicity mediated by HIV-1
Vpr. Expression in neural stem cells suppressed TNF-α expression and microglial activation, and improved
neurobehavioral deficits in mice. Collectively, these findings suggest a protective role for HERV-K duringpathological stress; this role may be the reason behind its conservation within the human genome.
Illumina Technology: Genome AnalyzerIIx

32

Microbes and metagenomics in human healthDennis F. E., Fujii Y., Haga K., Damanka S., Lartey B., et al. (2014) Identification of novel Ghanaian
G8P[6] human-bovine reassortant rotavirus strain by next generation sequencing. PLoS One 9: e100699
Group A rotaviruses are the leading cause of gastroenteritis in small children. The vaccine Rotarix was
introduced in Ghana in 2012, even though viral diversity was not available at the time. Genome sequencing
was performed in 2 G8P[6] Ghanaian strains from before vaccine introduction to obtain phylogeny anddiversity information. The authors show that both strains exhibited an unusual unreported genotype:
G8-P[6]-I2-R2-C2-M2-A2-N2-T2-E2-H3. Additionally, 10 out 11 genes were identical, with VP1 being theonly exception. Reassortment and transmission events with bovine/ovine/caprine rotaviruses were evident,
highlighting the need to monitor circulating animal and human strains that add diversity to Ghanaian rotavirus.
Illumina Technology: MiSeq Reagent Kit v2, MiSeq
Mohammadi P., di Iulio J., Munoz M., Martinez R., Bartha I., et al. (2014) Dynamics of HIV latency andreactivation in a primary CD4+ T cell model. PLoS Pathog 10: e1004156
HIV latency strategy allows the virus to prevent curation or being effectively purged from infected cells, evenwhen latency-reverting agents (eg, Vorinostat) inhibit histone deacetylases. A combination of RNA-Seq oflatently infected cells and a viral-encoded reporter were used in a CD4+ T-cell model to investigate thecharacteristics of latency and reactivation. The authors showed persistent presence of viral transcripts withvery limited translation during latency. Reactivating agents did increase viral transcription but failed to enhanceviral translation, suggesting that some posttranslational blocks not targeted by activating agents may lead to
HIV latency. These blocks need to be identified and addressed in order to clear cells from latent infection.

100. Parameswaran P., Sklan E., Wilkins C., Burgon
T., Samuel M. A., et al. (2010) Six RNA viruses
and forty-one hosts: viral small RNAs andmodulation of small RNA repertoires in vertebrate and invertebrate systems. PLoS Pathog
6: e1000764
101. van Mierlo J. T., Overheul G. J., Obadia B., vanCleef K. W., Webster C. L., et al. (2014) Novel
Drosophila viruses encode host-specific suppressors of RNAi. PLoS Pathog 10: e1004256
102. Hussain M. and Asgari S. (2014) MicroRNA-like viral small RNA from Dengue virus 2
autoregulates its replication in mosquito cells.
Proc Natl Acad Sci U S A 111: 2746-2751

Illumina Technology: TruSeq RNA Library Prep Kit, TruSeq Cluster Generation Kit, HiSeq 2000

Viral Small RNAs and Host Interactions
RNA viruses produce double-stranded RNA intermediates (virus-derived small RNA)
within cells and activate RNA interference (RNAi) mechanisms in insects and plants.
These antiviral responses seem linked to antiviral interferon responses. Cell-derived
small RNAs are, in turn, also disturbed during viral infections. However, some viruses
can interfere with these host cell responses by encoding suppressors of the RNAi
pathway.100, 101 Virus small RNAs could possibly have regulatory functions in viral
replication, as observed in Dengue virus-2.102

References
Jiang P., Zhou N., Chen X., Zhao X., Li D., et al. (2015) Integrative analysis of differentially expressed
microRNAs of pulmonary alveolar macrophages from piglets during H1N1 swine influenza A virus
infection. Sci Rep 5: 8167
H1N1 swine influenza A virus carries pandemic potential, and it also serves as a model to study pathogenesis
in humans. This study aimed to uncover differential expression of microRNA (miRNA) in pulmonary alveolar
macrophages of piglets infected with H1N1. The study showed that host miRNAs followed a downregulation
trend during the acute phase of infection that gradually returned to normal during disease recovery to avoid
severe lung damage. Additionally, miRNA-target regulatory networks could serve to identify functions andregulatory mechanisms of miRNAs during influenza infection.
Illumina Technology: HiSeq 2000, Genome AnalyzerIIx
Zhou Z., Li X., Liu J., Dong L., Chen Q., et al. (2015) Honeysuckle-encoded atypical microRNA2911
directly targets influenza A viruses. Cell Res 25: 39-49
Honeysuckle (HS, Lonicera japonica) is a traditional Chinese herb with reported activity against replicationof influenza virus. However, its specific mode of action has not been described. The authors used highthroughput sequencing to identify miRNA MIR2911 encoded by HS that targets a broad spectrum of Influenza
A viruses. MIR2911 was found in lungs and blood of mice given a HS decoction, suggesting it is heat-stable.
Synthetic and decoction preparations of M IR2911 inhibited PB2 and NS1 protein expression and also
replication of H1N1. The inhibitory effects of HS decoction were abolished by mutagenesis of PB2 and NS1,
and by anti-MIR2911 antagomir, suggesting that the concentration of MIR2911 in HS decoction was sufficient
to induce viral inhibitory effects. MIR2911 also inhibited H5N1 and H7N9 replication in vitro and in vivo. The
authors present the first active component identified in HS that effectively suppresses influenza virus A.
Illumina Technology: HiSeq 2000

An overview of recent publications featuring Illumina technology

33

Honeysuckle tea used in traditional Chinese medicine encodes a miRNA with inhibitory effects against
influenza A virus.
Cooper D. A., Jha B. K., Silverman R. H., Hesselberth J. R. and Barton D. J. (2014) Ribonuclease L
and metal-ion-independent endoribonuclease cleavage sites in host and viral RNAs. Nucleic Acids
Res 42: 5202-5216
Ribonuclease L (RNase L) is a metal-ion–independent endoribonuclease thought to contribute to immune
responses and cancer. However, little is known about the cellular and viral targets for this enzyme. The
authors validated 2’,3’-cyclic phosphate cDNA synthesis and Illumina sequencing methods to determine viraland cellular RNA targets for RNAse L. The study identified regions of hepatitis C virus and poliovirus RNA
genomes susceptible to RNAse L. The results also showed RNAse L-dependent and -independent cellulartargets within ribosomal RNAs and evidence of 2’,3’-cyclic phosphates at the ends of 5S rRNA.
Illumina Technology: MiSeq, Genome AnalyzerIIx
Li D. J., Verma D., Mosbruger T. and Swaminathan S. (2014) CTCF and Rad21 act as host cell
restriction factors for Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication by modulating
viral gene transcription. PLoS Pathog 10: e1003880
The mechanism for reactivation and lytic cycle of Kaposi’s sarcoma-associated herpes virus (KSHV) requires
transcription of about 80 lytic cycle genes and viral DNA replication. CTCF and cohesin are cellular proteinsthat may be involved in cycle regulation by binding to specific sites in herpes virus genomes, although themechanisms are not fully characterized. RNAi depletion of both CTCD and cohesin coupled to chromatin
immunoprecipitation sequencing (ChIP-Seq) showed that both proteins are restriction factors for viral
KSHV replication. Dissociation from the herpes viral genome led to increased viral yield. Both factors
initially activated transcription of the KSHV genome but later inhibited lytic transcription to prevent viral RNA
accumulation. The authors show that RNAi is an efficient way to knock down cellular proteins to evaluate theireffects in viral genomes.
Illumina Technology: ChIP-Seq DNA Library Prep Kit, TruSeq RNA Library Prep v2 Kit, HiSeq 2000
Shrinet J., Jain S., Jain J., Bhatnagar R. K. and Sunil S. (2014) Next generation sequencing reveals
regulation of distinct Aedes microRNAs during chikungunya virus development. PLoS Negl Trop Dis
8: e2616
The roles of virus-derived small RNAs (vsRNAs) on various cellular processes in insects has prompted
research on their role in disease transmission. This study focused on Aedes albopictus (Singh’s) cell line
carrying chikungunya virus (CHIKV). Compared to uninfected controls (n = 88), infected cells expressed 79
miRNAs. Closer analysis showed that 77 miRNAs common to both libraries were differentially expressed,
and that 8 specific miRNAs were altered by CHIKV infection by upregulation (n = 4) and downregulation (n= 4). Predicted targets for these miRNAs were clustered in 17 different pathways associated with immune
responses, amino acid degradation, and viral entry, among others. The authors provide pathways andinteractions for the differentially expressed miRNAs that may shed light on cellular modification exerted bymiRNAs during CHIKV infection.
Illumina Technology: TruSeq Small RNA Library Prep Kit, Genome AnalyzerIIx

34

Microbes and metagenomics in human healthAedes albopictus, Asian tiger mosquito.
Weng K. F., Hung C. T., Hsieh P. T., Li M. L., Chen G. W., et al. (2014) A cytoplasmic RNA virus
generates functional viral small RNAs and regulates viral IRES activity in mammalian cells. Nucleic
Acids Res 42: 12789-12805
The details of vsRNAs action against RNA viruses have not been explored in mammalian cells. NGS andNorthern blots identified 4 vsRNAs (1 through 4) in cells infected with enterovirus-71. vsRNA1 was highly
abundant in infected cells and associated with Dicer. vsRNA1 overexpression resulted in inhibition of viraltranslation and activity of internal ribosomal entry site (IRES) in infected cells. This mechanism may be
mediated by targeting the stem-loop II of the viral 5’-UTR that prevents interaction with IRES. Thus, vsRNA1
inhibition led to enhanced viral replication and protein synthesis. The authors describe a potential novel
mechanism for viral regulation mediated by interaction between vsRNA and IRES.

103. Lemey P., Rambaut A., Bedford T., Faria N.,
Bielejec F., et al. (2014) Unifying viral genetics
and human transportation data to predict the
global transmission dynamics of human influenza H3N2. PLoS Pathog 10: e1003932

Illumina Technology: HiSeq 2000

Human Viral Pathogens
Human incursion into wild habitats, climate change, international trade, globalization,and international travel are key factors in the emergence and transmission of viral
infectious diseases. Combining human mobility data and viral sequences proves how
these factors contribute to and help predict transmission dynamics, as demonstrated
in models for human influenza H3N2.103

References
Nordahl Petersen T., Rasmussen S., Hasman H., Caroe C., Baelum J., et al. (2015) Meta-genomic
analysis of toilet waste from long distance flights; a step towards global surveillance of infectious
diseases and antimicrobial resistance. Sci Rep 5: 11444
Globalization and massive transport systems contribute to the rapid spread of diseases around the world.
Monitoring worldwide transmission is a difficult task that can be helped by fast and reliable high-throughput
methods. The authors performed shotgun sequencing of toilet waste from 18 international flights arriving in
Copenhagen, Denmark from 9 cities in 3 regions of the world. Resistance genes against the antimicrobials
tetracycline, macrolides, and β-lactams were the most abundant in all samples. Flights originating in South
Asia contained higher abundance and diversity of these genes when compared to US flights. Norovirus andSalmonella enterica were also more abundant from South Asia, whereas C. difficile was more common in US
flights. This study shows the usefulness of shotgun sequencing for global surveillance, providing pathogen
detection, and monitoring antimicrobial resistance profiles.
Illumina Technology: HiSeq 2000

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Metagenomic surveys of toilet waste from air transport systems uncover the potential use of NGS inglobal health surveillance.
Briese T., Mishra N., Jain K., Zalmout I. S., Jabado O. J., et al. (2014) Middle East respiratory syndrome
coronavirus quasispecies that include homologues of human isolates revealed through whole-genome
analysis and virus cultured from dromedary camels in Saudi Arabia. MBio 5: e01146-01114
Antibodies to (and nucleic acids of) MERS-CoV have been identified in dromedaries, suggesting they
are the viral reservoirs of MERS-CoV human infections. In this study, whole virus genome sequenceswere recovered from nasal swabs of dromedaries in Saudi Arabia through direct sequencing. Dromedary
MERS-CoV consensus sequences were identical to human MERS-CoV sequences. More than 1 genomic
variant was found in dromedaries, implying that they can be infected by more than 1 strain of MERS-CoV,in contrast to single clonal sequences found in humans. These findings support the role of dromedaries
as reservoirs of MERS-CoV in human infections. Based on clonality differences, the authors suggest thepossibility that specific genotypes of MERS-CoV in dromedaries can pass bottleneck selection and achieveinterspecies transmission.
Illumina Technology: HiSeq 2500

Dromedaries may be the viral reservoirs of MERS-CoV human infections.
Brown B. A., Nix W. A., Sheth M., Frace M. and Oberste M. S. (2014) Seven Strains of Enterovirus
D68 Detected in the United States during the 2014 Severe Respiratory Disease Outbreak. Genome
Announc 2: e01201
Enterovirus D68 (EV-D68) was first identified in California in 1962, causing bronchiolitis and pneumonia.
Rarely detected for many years, it reappeared in 2009 and it is now causing the most widespread outbreakto date. Here, the CDC generated 7 genome sequences of cocirculating representative EV-D68 strains. The
authors described the genomic structure of these 7 strains and concluded that, based on their VP1 gene, thestrains are closely related to those previously isolated in the US, Europe, and Asia.
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36

Microbes and metagenomics in human healthChan B. K., Wilson T., Fischer K. F. and Kriesel J. D. (2014) Deep sequencing to identify the causes ofviral encephalitis. PLoS One 9: e93993
Clinical cases of encephalitis of unknown etiology abound and require non a priori techniques to determine
their etiology. To this end, 14 frozen normal and 7 frozen encephalitis brain samples were examined with deep
RNA sequencing and pathogen-specific PCR. Among encephalitis samples, measles and herpes simplex
virus type-1 sequences were found in a total of 2 and 3 brain samples, respectively. These results were inagreement with clinical and pathogen-specific PCR estimations, showing that metagenomic sequencing cancorrectly identify viral infections in frozen brain tissue.
Illumina Technology: TruSeq Library Prep Kit, HiSeq 2000
Escalera-Zamudio M., Nelson M. I., Cobian Guemes A. G., Lopez-Martinez I., Cruz-Ortiz N., et al.
(2014) Molecular epidemiology of influenza A/H3N2 viruses circulating in Mexico from 2003 to 2012.
PLoS One 9: e102453
The H1N1 2009 influenza pandemic highlights the need to address the nature of viral reservoirs throughmolecular epidemiology approaches. The authors sequenced 19 influenza A/H3N2 strains isolated in Mexico
between 2003 and 2012, analyzed their phylogenetic relationships, and compared antigenic determination.
The study showed that many lineages cocirculate within the same flu season. Some of these lineages
persist across different seasons, becoming possible reservoirs for reassortment events. Phylogenetic
characterization did not necessarily correlate with antigenic identity, underscoring the need to use genomic
determination tools, in addition to antigenic data, for surveillance of influenza virus.
Illumina Technology: Genome AnalyzerIIx
Peng X., Alfoldi J., Gori K., Eisfeld A. J., Tyler S. R., et al. (2014) The draft genome sequence of the ferret(Mustela putorius furo) facilitates study of human respiratory disease. Nat Biotechnol 32: 1250-1255
The domestic ferret is considered the “gold standard” model for human influenza virus infection andtransmission; however, its full genome has not been established, limiting inferences based on this animalmodel. This study obtained the draft genome sequence of 1 female ferret, selected for its low heterozygosity,and then determined the total RNA sequence of different tissues obtained from 24 ferrets. Similarly,transcriptomic profiles in 42 ferrets infected with 1918 and 2009 pandemic influenza strains showed different
transcriptomic signatures in lung and trachea tissues. Microarray data from 16 ferret samples reflecting CF
disease progression showed that CFTR knockout animals have pathways of disease that go undetected ininfant samples.
Illumina Technology: HiSeq 100 bp PE reads
Sikora D., Rocheleau L., Brown E. G. and Pelchat M. (2014) Deep sequencing reveals the eight facetsof the influenza A/HongKong/1/1968 (H3N2) virus cap-snatching process. Sci Rep 4: 6181
NAs and uses these capped fragments as primers for viral mRNA synthesis in a process called “capsnatching.” The authors deep sequenced all the 5’ ends of viral mRNAs in human and mouse cells infectedwith influenza A/HongKong/1/1968 (H3N2). These regions are variable in length, motif nucleotides, andhijacked host mRNAs. Mapping the reads to known transcription start sites showed that the virus targets
the most abundant cellular mRNAs. Overall findings suggest that viral polymerase-viral mRNA complexes
compete minimally for host mRNA targets. Other details about cap snatching are also provided.
Illumina Technology: HiSeq 2000
Wylie K. M., Wylie T. N., Orvedahl A., Buller R. S., Herter B. N., et al. (2015) Genome sequence ofenterovirus D68 from St. Louis, Missouri, USA. Emerg Infect Dis 21: 184-186
As of October 2014, CDC had confirmed 594 cases of human EV-D68 in 43 states, an unprecedented level
for this particular virus. Only 5 viral sequences were available until 2014, limiting molecular epidemiology
studies. The authors of this study provide 1 full genome and 8 partial sequences of clinical strains of EV-D68
isolated in St. Louis. These sequences were compared to other 7 sequences from the Midwest generated
by the CDC. All the St. Louis sequences were highly similar, sharing up to 99% nucleotide identity. The full
St. Louis genome was similar to the Midwest viruses, and they clustered together with European and Asianisolates from the past several years. This sequence is another tool for genomic comparison of this rapidly
spreading virus, although detailed characterization would be useful to associate genomic changes withphenotypic traits.
Illumina Technology: HiSeq 2500 100 bp PE reads

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EbolaOutbreaks of EBOV, influenza virus, MERS-CoV, and Chikunguya virus exemplify thecomplex social and scientific challenges posed by emergent pathogens. Rapid andreliable tools for viral identification are required to monitor outbreaks in support ofepidemiological studies nearly in real-time.104 NGS has become a powerful tool thatmeets these demands.105 Its use in metagenomic analysis of toilet waste from longdistance flights illustrates its potential use in global health and threat surveillance.106

References
Carroll M. W., Matthews D. A., Hiscox J. A., Elmore M. J., Pollakis G., et al. (2015) Temporal andspatial analysis of the 2014-2015 Ebola virus outbreak in West Africa. Nature 524: 97-101
Complementing multiple efforts to support epidemic studies of the current EBOV outbreak, this study resulted
in deep sequencing of 179 samples processed by the European Mobile Laboratory in Guinea to reveal an
epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. The analysis
concluded that EBOV was introduced from Guinea into Sierra Leone, most likely in April or early May 2014.
Sequences corresponding to August, September, and October 2014 indicated that this lineage evolved
independently in Guinea. Along with similar efforts in Sierra Leone, these studies inform the effectiveness ofcontrol measures and describe the ongoing history of the outbreak.
Illumina Technology: ScriptSeq v2 RNA-Seq Library Prep Kit, HiSeq 2500 v4 125bp PE reads
Park D. J., Dudas G., Wohl S., Goba A., Whitmer S. L., et al. (2015) Ebola Virus Epidemiology,
Transmission, and Evolution during Seven Months in Sierra Leone. Cell 161: 1516-1526
The Makona variant of EBOV continues to cause damage in African countries. Insight into virus transmission
and dynamics are important to create intervention measures. The authors sequenced 232 new genomes thatwere sampled over 7 months in Sierra Leone and compared them to data obtained earlier in the epidemic.
Sustained human-to-human transmission within Sierra Leone was confirmed by this analysis, without
evidence of import of EBOV across borders after initial introduction. The emergence of intrahost genetic
variants, host-to-host transmission, effective purifying selection suppression of nonsynonymous mutations,and changes in the mucin-like domain over this longer timescale were detected. Importantly, the authors
noted that the estimated viral evolution rate was lower and closer to the long-term rate than to the rateestimated early in the outbreak. This study provides insightful details about epidemic dynamics in a longer
timescale and highlights the importance of continued sampling and sequencing throughout the epidemic.
Illumina Technology: Nextera XT DNA Library Prep Kit, HiSeq 2500 100 bp PE reads
Simon-Loriere E., Faye O., Faye O., Koivogui L., Magassouba N., et al. (2015) Distinct lineages ofEbola virus in Guinea during the 2014 West African epidemic. Nature 524: 102-104
The authors sequenced 85 EBOV samples from patients infected from July to November 2014 in Guinea.
Sequence analysis showed sustained transmission and cocirculation of 3 viral lineages: One was unique
to Guinea and closely related to the earliest sampled viruses; the second contained viruses probably
reintroduced from Sierra Leone; and the third was later spread to Mali. Each lineage contained specific setsof mutations, including the mucin-like domain of the viral glycoprotein. The authors discuss the implication of
glycoprotein mutations in phenotypic variation.
Illumina Technology: ScriptSeq v2 RNA-Seq Library Prep Kit, HiSeq 2500 v4 125 bp PE reads

Ebola virus.

38

Microbes and metagenomics in human health104. Kugelman J. R., Wiley M. R., Mate S., Ladner
J. T., Beitzel B., et al. (2015) Monitoring of
Ebola Virus Makona Evolution through Establishment of Advanced Genomic Capability in
Liberia. Emerg Infect Dis 21: 1135-1143
105. Lipkin W. I. (2013) The changing face of
pathogen discovery and surveillance. Nat Rev
Microbiol 11: 133-141
106. Nordahl Petersen T., Rasmussen S., Hasman
H., Caroe C., Baelum J., et al. (2015) Meta-genomic analysis of toilet waste from long
distance flights; a step towards global surveillance of infectious diseases and antimicrobial
resistance. Sci Rep 5: 11444

Zaraket H., Baranovich T., Kaplan B. S., Carter R., Song M. S., et al. (2015) Mammalian adaptation of
influenza A(H7N9) virus is limited by a narrow genetic bottleneck. Nat Commun 6: 6553
Human infection with avian influenza A (H7N9) virus is mostly associated with contact with infected poultry,
although transmission between humans in limited. This study reported that H7N9 was highly diverse andasymptomatic in poultry. In ferrets, the opposite occurred: diversity was highly restricted and was not fullytransmissible among these species. A series of mutations in different viral genes were associated with ferret
infection, but they came with a fitness cost that limited further host-to-host transmission. This mechanism
limits species jumps and provides a tool for risk assessment in pandemic preparedness.
Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq 150 bp PE reads
Gire S. K., Goba A., Andersen K. G., Sealfon R. S., Park D. J., et al. (2014) Genomic surveillanceelucidates Ebola virus origin and transmission during the 2014 outbreak. Science 345: 1369-1372
The authors were able to recover genome sequences in the early stages of the African Ebola epidemic. A
total of 99 EBOV genomes from 78 patients in Sierra Leone were generated using NGS. These genomes
showed accumulated intrahost and interhost genetic variation. The results showed that this West Africa
variant probably diverged from central African lineages around 2004 and sustained human-to-humantransmission upon entering Guinea in May 2004. The authors point out that many viral genes show distinct
mutations that should be monitored to evaluate their impact on intervention measures.
Illumina Technology: Hybridase Thermostable RNase H, Nextera XT DNA Library Prep Kit, HiSeq 100 bp
PE reads

HIV107. Elliott J. H., Wightman F., Solomon A., Ghneim
K., Ahlers J., et al. (2014) Activation of HIV
transcription with short-course vorinostat in
HIV-infected patients on suppressive antiretroviral therapy. PLoS Pathog 10: e1004473
108. Mascola J. R. (2015) HIV. The modern era of
HIV-1 vaccine development. Science 349:
139-140
109. Kouri V., Khouri R., Aleman Y., Abrahantes Y.,
Vercauteren J., et al. (2015) CRF19_cpx is an
Evolutionary fit HIV-1 Variant Strongly Associated With Rapid Progression to AIDS in Cuba.
EBioMedicine 2: 244-254
110. Dillon S. M., Lee E. J., Kotter C. V., Austin G.
L., Dong Z., et al. (2014) An altered intestinal
mucosal microbiome in HIV-1 infection is associated with mucosal and systemic immune
activation and endotoxemia. Mucosal Immunol
7: 983-994
111. Mutlu E. A., Keshavarzian A., Losurdo J.,
Swanson G., Siewe B., et al. (2014) A compositional look at the human gastrointestinal
microbiome and immune activation parameters in HIV infected subjects. PLoS Pathog 10:
e1003829

Viruses like HIV-1 continue to cause concern due to their ability to establish latency
and evade therapy.107, 108 There is also the emergence of aggressive variants that
progress quickly to AIDS.109 The use of NGS in HIV research has shown viral
contribution to dysbiosis of the intestinal microbiome, shifting toward increased
abundance of Prevotella, association with immune activation in nontreatedpatients,110 and in cohorts treated with antiretroviral therapy (ART).111

References
Cotten M., Oude Munnink B., Canuti M., Deijs M., Watson S. J., et al. (2014) Full genome virus
detection in fecal samples using sensitive nucleic acid preparation, deep sequencing, and a novel
iterative sequence classification algorithm. PLoS One 9: e93269
Metagenomic sequencing of fecal viromes can provide clues about pathogens in a sample without a priori
assumptions about its identity. The authors set out to develop a full-genome detection process by combining
nucleic acid extraction, library preparation, and virus identification using Illumina sequencing and bioinformaticalgorithms. De novo assembly was used to generate full viral genomes. The procedure was tested in fecal
samples from patients infected with HIV-1. This cohort exhibited an array of viruses, yielding 12 complete
viral genomes from 6 virus families, including opportunistic enteropathic viruses. This method is useful in viraldetection and may improve the analysis of changes associated with HIV-1 progression.
Illumina Technology: MiSeq
Dillon S. M., Lee E. J., Kotter C. V., Austin G. L., Dong Z., et al. (2014) An altered intestinal mucosal
microbiome in HIV-1 infection is associated with mucosal and systemic immune activation andendotoxemia. Mucosal Immunol 7: 983-994
HIV-1 infection is known to cause intestinal immune system disturbance, leading to microbial translocation,
lipopolysaccharide (LPS) leakage, and systemic activation of the immune system. 16S V4 rRNA gene
amplicon sequencing of colon biopsies was used to assess gut microbiota changes. The authors showed
gut microbiota shifts in HIV-1–infected individuals. Greater abundance of Prevotella species and decreased
numbers of Bacteroides suggested disruption of the gut microbial community, which was accompanied by
activated colonic T cells and dendritic cells, microbial translocation, and blood T-cell activation. Thus, HIV-1
infections lead to gut microbiota disruption and downstream effects in local and systemic immune activation.
Illumina Technology: MiSeq Reagent Kit v2
Dudley D. M., Bailey A. L., Mehta S. H., Hughes A. L., Kirk G. D., et al. (2014) Cross-clade
simultaneous HIV drug resistance genotyping for reverse transcriptase, protease, and integrase
inhibitor mutations by Illumina MiSeq. Retrovirology 11: 122
Viral resistance to ART commonly arises in the pol gene of HIV-1 virus. Current genotyping methods are
optimized for subtype B virus and identify resistance to protease and reverse transcriptase inhibitors, but not
to integrase inhibitors. The authors developed a universal primer set targeting the pol gene of M HIV subtypes

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39

that, when coupled to Illumina MiSeq sequencing, could detect resistance against the 3 types of antivirals.
This genotyping method was tested on 62 samples, being consistent and sensitive at detecting drug
resistance mutations. This tool could aid in quick detection and tracking of antiviral resistance variants.
Illumina Technology: Nextera XT DNA Library Prep, MiSeq

PhagesBacteriophages represent an absolute majority of all organisms in the biosphere.112
These organisms are viruses that infect bacteria and play a prominent role in shaping
microbial populations.113 The genetic diversity of the population is very high, andit appears that phages have been actively evolving for billions of years. Frequenthorizontal genetic exchange among phages results in pervasive mosaicism intheir architectures and the emergence of novel bacterial pathogens.114 The current
crisis with antibiotic-resistant bacteria has renewed interest in phage therapy andbiocontrol approaches in infection control.115

112. Hatfull G. F. and Hendrix R. W. (2011) Bacteriophages and their genomes. Curr Opin Virol
1: 298-303
113. Reyes A., Semenkovich N. P., Whiteson K.,
Rohwer F. and Gordon J. I. (2012) Going viral:
next-generation sequencing applied to phage
populations in the human gut. Nat Rev Microbiol 10: 607-617
114. Muniesa M., Hammerl J. A., Hertwig S., Appel
B. and Brussow H. (2012) Shiga toxin-producing Escherichia coli O104:H4: a new challenge
for microbiology. Appl Environ Microbiol 78:
4065-4073
115. Fernandes P. (2006) Antibacterial discovery
and development--the failure of success? Nat
Biotechnol 24: 1497-1503
116. Liu J., Yan R., Zhong Q., Ngo S., Bangayan N.
J., et al. (2015) The diversity and host interactions of Propionibacterium acnes bacteriophages on human skin. ISME J 9: 2078-2093
117. Jensen K. C., Hair B. B., Wienclaw T. M., Murdock M. H., Hatch J. B., et al. (2015) Isolationand Host Range of Bacteriophage with Lytic
Activity against Methicillin-Resistant Staphylococcus aureus and Potential Use as a Fomite
Decontaminant. PLoS One 10: e0131714
118. Cowley L. A., Beckett S. J., Chase-Topping
M., Perry N., Dallman T. J., et al. (2015)
Analysis of whole genome sequencing for the
Escherichia coli O157:H7 typing phages. BMC
Genomics 16: 271
119. Waldron D. (2015) Microbial ecology: Sortingout viral dark matter. Nat Rev Microbiol 13:
526-527120. Waller A. S., Yamada T., Kristensen D. M.,
Kultima J. R., Sunagawa S., et al. (2014) Classification and quantification of bacteriophagetaxa in human gut metagenomes. ISME J 8:
1391-1402

Bacteriophage.

Bacteria are equipped to resist phage infection by encoding CRISPR sequences,although resistance is variable because it occurs in P. acnes strains in human skin.116 The
use of phages as fomite decontamination agents against MRSA 117 exemplifies promisingalternatives to antimicrobial use. In addition, phage typing of pathogenic bacteria is a
resource for surveillance and epidemiologic investigation of enteropathogen outbreaks.118
The bacterial virome may have a profound impact in health and the environment.119
An average of 5 bacteriophage taxa exist in variable abundance in the human gut.
Some of these taxa are characteristic of specific human populations, confirming
specific host-phage associations.120 Changes in signature viromes could be earlymarkers of disturbance and could provide noninvasive diagnostic tests in the future.
Virophages are satellite viruses that inhibit or impair the reproduction of the auxiliary
virus. Virophage sequences are globally distributed, including the human 121 andanimal gut.122 Their role is still largely unexplored.

Reviews
Krupovic M. and Koonin E. V. (2015) Polintons: a hotbed of eukaryotic virus, transposon and plasmid
evolution. Nat Rev Microbiol 13: 105-11540

Microbes and metagenomics in human health121. Zhou J., Zhang W., Yan S., Xiao J., Zhang Y.,
et al. (2013) Diversity of virophages in metagenomic data sets. J Virol 87: 4225-4236
122. Yutin N., Kapitonov V. V. and Koonin E. V.
(2015) A new family of hybrid virophages froman animal gut metagenome. Biol Direct 10: 19

References
Cowley L. A., Beckett S. J., Chase-Topping M., Perry N., Dallman T. J., et al. (2015) Analysis of wholegenome sequencing for the Escherichia coli O157:H7 typing phages. BMC Genomics 16: 271
E. coli O157:H7 produces hemolytic uremic syndrome and bloody diarrhea. Phage typing helps public healthsurveillance efforts, given that certain phages associate with niches, age groups, and severity. The authors
sequenced 16 typing phages (T4 and T7) and showed that T7 phages differ in only 3 nucleotides whereas T4
phages fall into 3 different groups of similar genomic sequences.
Illumina Technology: TruSeq DNA Library Prep Kit, MiSeq 150 bp PE reads, Nextera DNA Library Prep Kit,
Genome AnalyzerIIx

123. World Health Organization (2015) Smallpox.
124. Wassilak S. G., Oberste M. S., Tangermann
R. H., Diop O. M., Jafari H. S., et al. (2014)
Progress toward global interruption of wild
poliovirus transmission, 2010-2013, and tackling the challenges to complete eradication. J
Infect Dis 210 Suppl 1: S5-15

Jakhetia R. and Verma N. K. (2015) Identification and Molecular Characterisation of a Novel Mu-Like
Bacteriophage, SfMu, of Shigella flexneri. PLoS One 10: e0124053
S. flexneri is a common cause of dysentery in developing countries, and several of its phages have been
described. The authors characterized the genomic features and phenotypic characteristics of the Mu-likephage SfMu by NGS. O antigen was identified as a SfMu receptor. Prophages and remnants were foundin different S. flexneri serotypes, indicating that such transposable elements may be common in S. flexneri.
Understanding phage-bacteria interactions will help to elucidate their roles in evolution and pathogenesis.
Illumina Technology: MiSeq 250 bp PE reads
Moon B. Y., Park J. Y., Hwang S. Y., Robinson D. A., Thomas J. C., et al. (2015) Phage-mediatedhorizontal transfer of a Staphylococcus aureus virulence-associated genomic island. Sci Rep 5: 9784
The pathogenic island vSaβ, found in almost all Staphylococcus aureus strains, differs greatly in virulencegene content. Diversity and mobilization of the island remain poorly understood. In this study, vSaβ and an
associated prophage were sequenced and characterized. The mobility of vSaβ to human and animal S. aureus
is mediated by this prophage through multiple conversions of transducible particles that may also be involved
in diversity acquisition. These findings highlight the role of phages in the pathogenic evolution of S. aureus.
Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq v2
Sangster W., Hegarty J. P. and Stewart D. B., Sr. (2015) Phage tail-like particles kill Clostridium difficile
and represent an alternative to conventional antibiotics. Surgery 157: 96-103
Recurrent C. difficile infections (CDI) are difficult to control with antibiotic courses. As a result, alternative
therapies like phage tail-like particles (PTLP)—proteins morphologically similar to phages and produced by
C. difficile—are being explored. Purified PTLP from a clinical C. difficile sample was tested against patientderived C. difficile of varying ribotypes. The PTLP genomic cluster was sequenced and annotated. The
bactericidal activity of PTLP was ribotype- and species-dependent, and the putative genes required for killingwere identified. These in vitro results are promising for the development of alternative therapies against CDI.
Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq 150 bp PE reads
Hare J. M., Ferrell J. C., Witkowski T. A. and Grice A. N. (2014) Prophage induction and differential
RecA and UmuDAb transcriptome regulation in the DNA damage responses of Acinetobacterbaumannii and Acinetobacter baylyi. PLoS One 9: e93861
The SOS response requires RecA to relieve LexA transcriptional repression. In Acinetobacter species,
umuDAb is instead required for induction after DNA damage, suggesting it might be a LexA analog. RNASeq defined the DNA damage transcriptome of WT, recA, and umuDAb mutant strains of A. baylyi and
A. baumannii, which had differential reliance on recA- and umuDAb-induced SOS genes. A. baumannii
genes were clustered in 3 prophage regions, and bacteriophage particles were observed after mitomycin C
treatment. These results suggest that both species possess a robust and complex DNA damage response
involving both recA-dependent and recA-independent regulons. They also demonstrate that, althoughumuDAb has a specialized role in repressing error-prone polymerases, additional regulators participate inthese species’ transcriptional response to DNA damage.
Illumina Technology: Genome AnalyzerIIx

Virus Vaccine Development
Smallpox was successfully eradicated through massive immunization programs in the
1980s.123 Substantial progress has since been made toward poliovirus eradication,
despite the presence of WT poliovirus pockets in a few countries.124 After polio
virus, human enterovirus 71 (HEV71) is the most threatening neurotropic enterovirus.

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41

Effective vaccines remain unavailable despite its recent emergence in the Asia-Pacificregion,125 a situation shared with other serious emerging pathogens, like SARS.
A different picture has characterized the search for an HIV-1 vaccine. The large
efforts devoted to vaccine development against HIV-1 have yielded only modest
success, due to factors such as latency and antigenic variation.126, 127 Paradoxically,
technological improvements from these efforts have boosted other vaccine
development programs. For example, engineered cytokine-like adjuvants attached
to virus-like particles (VLPs) improved immunogenicity in HIV and have been applied
to other pathogens.128 Some cancers have been associated with virus infection. The
viral sequences of HPV-18 have been identified in ovarian cancer samples, implyingthat current vaccine strategies that include this strain might help to reduce thenumber of cases of this type of neoplasia.129
Many issues are inherent to vaccine development. For example, WT strains areable to replace vaccine strains over time, and genetic instability has been reported.
These cases impact vaccine effectiveness and make genetic monitoring of live andattenuated vaccines an important tool in immunization strategies.130

Reviews
Bande F., Arshad S. S., Bejo M. H., Moeini H. and Omar A. R. (2015) Progress and challenges toward thedevelopment of vaccines against avian infectious bronchitis. J Immunol Res 2015: 424860
Beasley D. W., McAuley A. J. and Bente D. A. (2015) Yellow fever virus: genetic and phenotypic diversity andimplications for detection, prevention and therapy. Antiviral Res 115: 48-70
Salazar-Gonzalez J. A., Angulo C. and Rosales-Mendoza S. (2015) Chikungunya virus vaccines: Current
strategies and prospects for developing plant-made vaccines. Vaccine 33: 3650-3658
Sanchez-Sampedro L., Perdiguero B., Mejias-Perez E., Garcia-Arriaza J., Di Pilato M., et al. (2015) The
evolution of poxvirus vaccines. Viruses 7: 1726-1803
Archin N. M., Sung J. M., Garrido C., Soriano-Sarabia N. and Margolis D. M. (2014) Eradicating HIV-1
infection: seeking to clear a persistent pathogen. Nat Rev Microbiol 12: 750-764

References
Fulton B. O., Sachs D., Beaty S. M., Won S. T., Lee B., et al. (2015) Mutational Analysis of Measles
Virus Suggests Constraints on Antigenic Variation of the Glycoproteins. Cell Rep 11: 1331-1338
The mechanisms behind measles virus (MeV) antigenic stability have not been fully explored. In this study,
MeV was subjected to transposon insertional mutagenesis, and viruses that tolerated insertions were rescued.
NGS was used to identify insertion sites and the evenness of the MeV transposon library. Insertions in theglycoproteins of MeV (F and H proteins) were severely underrepresented when compared to previous work onother viruses, such as influenza. This study shows that changes in F and H glycoproteins of MeV are not welltolerated, limiting the sequence variation required to escape antibody neutralization. This finding allows fordurable immunity after infection and vaccination.
Illumina Technology: TruSeq DNA LT Library Prep Kit, HiSeq 2000 100 bp SE reads
Marsh A. K., Ambagala A. P., Perciani C. T., Russell J. N., Chan J. K., et al. (2015) Examining thespecies-specificity of rhesus macaque cytomegalovirus (RhCMV) in cynomolgus macaques. PLoS
One 10: e0121339
Cytomegalovirus (CMV) is highly species-specific, which may be a limiting factor for its use as a vaccine
vector. In this study, cynomolgus macaques were infected with a recombinant, laboratory-adapted rhesus
macaque cytomegalovirus (RhCMV). Sequencing was used to determine the viral genetic integrity andenhanced green fluorescent protein (eGFP) insertion site. Infected macaques did not show signs of disease,42

Microbes and metagenomics in human health125. Kok C. C. (2015) Therapeutic and prevention
strategies against human enterovirus 71 infection. World J Virol 4: 78-95
126. Mascola J. R. (2015) HIV. The modern era of
HIV-1 vaccine development. Science 349:
139-140
127. Young B. E., Sadarangani S. P. and Leo Y. S.
(2015) The avian influenza vaccine Emerflu.
Why did it fail? Expert Rev Vaccines 14:1125-1134
128. Feng H., Zhang H., Deng J., Wang L., He Y.,
et al. (2015) Incorporation of a GPI-anchoredengineered cytokine as a molecular adjuvant
enhances the immunogenicity of HIV VLPs. Sci
Rep 5: 11856
129. Roos P., Orlando P. A., Fagerstrom R. M. andPepper J. W. (2015) In North America, someovarian cancers express the oncogenes of
preventable human papillomavirus HPV-18. Sci
Rep 5: 8645
130. Gladstone R. A., Jefferies J. M., Tocheva A. S.,
Beard K. R., Garley D., et al. (2015) Five winters of pneumococcal serotype replacementin UK carriage following PCV introduction.
Vaccine 33: 2015-2021

presence of the virus in tissue samples, or eGFP expression. Collectively, the data suggest that cynomolgus
were not productively infected with RhCMV. The authors discuss the use of lab-adapted and primary virusesin vaccine development.
Illumina Technology: MiSeq 150 bp PE reads
Mee E. T., Minor P. D. and Martin J. (2015) High resolution identity testing of inactivated poliovirus
vaccines. Vaccine 33: 3533-3541
Quality control is a critical component of vaccine development. Thus, reliable methods are required to identify
vaccine strains and distinguish biological contaminants. Random RT-PCR and high-throughput sequencing
were used to recover full-length genomes from monovalent and trivalent poliovirus vaccines at various
stages of manufacturing. The method correctly identified expected strains, discriminating them from Sabin
1 and Mahoney strains. Additionally, slight variations can be introduced to characterize minor variants anddistinguish closely related products.
Illumina Technology: Nextera XT Library Prep Kit, MiSeq v2 250 bp PE reads

Poliovirus capsid.
Ang L., Arboleya S., Lihua G., Chuihui Y., Nan Q., et al. (2014) The establishment of the infant intestinal
microbiome is not affected by rotavirus vaccination. Sci Rep 4: 7417
The effects of oral vaccination against rotavirus on gut microbiota establishment in 3 infants was assessed
through metagenomics, PCR-denaturing gel gradient electrophoresis (DGGE), and short chain fatty acids
analyses of prevaccination and postvaccination fecal samples. Microbiomes were similar at the functional
level, and vaccination did not seem to have a detectable effect in the establishment of gut microbiota.
Illumina Technology: HiSeq 2000 100 bp PE reads, CASAVA v1.8.2
Beck A., Tesh R. B., Wood T. G., Widen S. G., Ryman K. D., et al. (2014) Comparison of thelive attenuated yellow fever vaccine 17D-204 strain to its virulent parental strain Asibi by deep
sequencing. J Infect Dis 209: 334-344
The characterization of attenuated live vaccines, relative to their parental strains, reveals the mechanisms ofviral attenuation, vaccine stability, and the risk of reversion to virulence. The authors applied deep sequencing
to a live attenuated yellow fever virus strain, 17D-204, and its parental strain, Asibi. They found lack ofquasispecies diversity and discrete attenuating mutations in the vaccine strain, features that might correlate in
the attenuation of live viral vaccines.
Illumina Technology: HiSeq 1000 50 bp PE reads
Zhou W., Gao S., Podgorska K., Stadejek T., Qiu H. J., et al. (2014) Rovac is the possible ancestorof the Russian lapinized vaccines LK-VNIVViM and CS strains but not the Chinese strain (C-strain)
vaccine against classical swine fever. Vaccine 32: 6639-6642
Lapinized vaccines are attenuated by passing through rabbits. The origins of lapinized vaccines against
classical swine fever virus (CSFV) are lost in history. The authors performed NGS to elucidate vaccine origins
by phylogenetic analysis of the historical Russian Rovac strain and its lapinized vaccines, LK-VNIVViM and
CS. They also compared it to the lapinized Chinese strain (C-strain). Genetic analysis confirmed a close
relationship between the Russian strains Rovac and LK-VNIVViM, and showed how the latter had an ancestryrole in relation to the CS strain and its derivative, RUCSFPLUM. The authors suggest that the Rovac vaccine
is the possible ancestor of the Russian vaccine strains but not the C-strain vaccine.
Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq

An overview of recent publications featuring Illumina technology

43

MICROBIAL PATHOGENESISOf the vast number of environmental microbes, a very small percentage cause
infectious diseases. Microbial genomes are dynamic and can employ processes
such as horizontal gene transfer, SNP, gene duplication, gene loss, and others.
Bacterial pathogenomics offers snapshots of these constant changes.

131

Culture and isolation methods require a priori knowledge about the identity of thecausative agents. NGS does not require assumptions about microbial identity andallows functional inferences about the microorganism(s) in question, uncovering aspectsof pathogen biology and evolution. These advantages are fundamental in understanding
pathogenesis. They can support epidemiological studies, identify public health threats,and detect the emergence of strains not controlled by existing vaccines.132

Reviews
Land M., Hauser L., Jun S. R., Nookaew I., Leuze M. R., et al. (2015) Insights from 20 years of bacterial
genome sequencing. Funct Integr Genomics 15: 141-161
Angelakis E. and Raoult D. (2014) Methods for the discovery of emerging pathogens. Microb Pathog 77: 114-118

References
Bruggemann H., Brzuszkiewicz E., Chapeton-Montes D., Plourde L., Speck D., et al. (2015) Genomicsof Clostridium tetani. Res Microbiol 166: 326-331
Only 2 genomes of C. tetani are available. The authors used NGS to obtain the genomes of 3 additional
C. tetani strains and compared them with the 2 previously sequenced genomes. Major features include
tetanus-toxin–encoding plasmids and a core genome that contains about 85% of all genes. The non–core
chromosome includes elements for environmental interaction, defenses, and fitness functions.
Illumina Technology: Genome AnalyzerIIx
Guerra-Assuncao J. A., Houben R. M., Crampin A. C., Mzembe T., Mallard K., et al. (2015) Recurrence
due to relapse or reinfection with Mycobacterium tuberculosis: a whole-genome sequencing
approach in a large, population-based cohort with a high HIV infection prevalence and active followup. J Infect Dis 211: 1154-1163
Tuberculosis (TB) recurrence may be due to relapse with the original strain or reinfection with a new strain.
WGS was performed in all available cultures from a TB cohort actively followed after end of treatment innorthern Malawi. Relapse was defined by a difference of ≤ 10 SNPs, while reinfections had a differenceof ≥ 100 SNPs. Reinfection was associated with HIV infection, while relapses associated with lineage-3
strains and isoniazid resistance. Several factors contribute to TB recurrence; however, differences in the M.
tuberculosis genome also play a role in relapse and infection.
Illumina Technology: HiSeq 2000 100 bp PE reads
Springer D. J., Billmyre R. B., Filler E. E., Voelz K., Pursall R., et al. (2014) Cryptococcus gattii VGIII
isolates causing infections in HIV/AIDS patients in Southern California: identification of the local
environmental source as arboreal. PLoS Pathog 10: e1004285
The C. gatii fungal pathogen affects immunocompromised patients. Ongoing outbreaks in the US Northwest
have called attention to the need for finding a link between clinical isolates and environmental sources in
California. The authors isolated active C. gatii VGIII from soil debris near residences of infected patients.
Multilocus sequence typing (MLST) and WGS showed that these environmental isolates were the source ofhuman infections. This study highlights that environmental sources present a risk of C. gatii infection in HIV
patients, a risk that has previously been underestimated.
Illumina Technology: HiSeq 2500 100 bp PE reads

44

Microbes and metagenomics in human health131. Pallen M. J. and Wren B. W. (2007) Bacterial
pathogenomics. Nature 449: 835-842
132. Peacock S. (2014) Health care: Bring microbial
sequencing to hospitals. Nature 509: 557-559

Important Microorganisms in Human HealthSome microorganisms have a long history of causing infections in humans andanimals,133 whereas some bacteria are host-specific and others have a wide rangeof tropism.134 However, changes in lifestyle, globalization,135 vaccination, and the riseof antibiotic resistance are redefining the landscape of those microorganisms weconsider important. The molecular mechanisms of disease are rooted in the genome,
but interactions between microbial communities and their changing ecosystems areimportant drivers in the evolution of bacterial transmission and pathogenesis.136, 137, 138

Mycobacteria
Slow-growing mycobacteria are responsible for a wide range of diseases in humanand animal hosts (Table 2).

Table 2: Mycobacteria and Diseases.

Name

Host

DiseaseReference

Mycobacterium abscessus

HumansA major pathogen in
CF patients; has been
associated with poor
clinical outcomes

139

Mycobacterium abscessus
subsp. massiliense

Humans

CF patients and soft-tissue
infections

140

Mycobacterium avium
Subsp. hominissuis

Pigs, humans

Disseminated disease inimmunocompromised
hosts, such as individuals
infected by HIV or
pulmonary disease inindividuals without systemic
immunosuppression

141

Mycobacterium lepromatosis

HumansDiffuse lepromatous
leprosy and a reactional
state known as Lucio’s
phenomenon

142

Mycobacterium terrae
complex

HumansRare but severe pulmonary
disease143

Mycobacterium tuberculosis

HumansTuberculosis

144

Mycobacterium ulcerans

HumansBuruli ulcer, an ulcerative
skin infection

145

The M. tuberculosis Beijing lineage is causing a global TB epidemic, often found withincreased levels of drug resistance. WGS is a tool to analyze the evolution of thispathogen, support epidemiological studies that place isolates in a global context,146
and rapidly determine some drug resistance patterns.147

133. Kay G. L., Sergeant M. J., Zhou Z., Chan J.
Z., Millard A., et al. (2015) Eighteenth-century
genomes show that mixed infections werecommon at time of peak tuberculosis in
Europe. Nat Commun 6: 6717
134. Bos K. I., Harkins K. M., Herbig A., Coscolla
M., Weber N., et al. (2014) Pre-Columbian mycobacterial genomes reveal seals as a sourceof New World human tuberculosis. Nature
514: 494-497
135. Petty N. K., Ben Zakour N. L., Stanton-Cook
M., Skippington E., Totsika M., et al. (2014)
Global dissemination of a multidrug resistantEscherichia coli clone. Proc Natl Acad Sci U S
A 111: 5694-5699
136. Wilson D. J. (2012) Insights from genomics
into bacterial pathogen populations. PLoS
Pathog 8: e1002874
137. Ochman H. and Moran N. A. (2001) Genes
Lost and Genes Found: Evolution of Bacterial
Pathogenesis and Symbiosis. Science 292:
1096-1099
138. van Boven M., Mooi F. R., Schellekens J. F.,
de Melker H. E. and Kretzschmar M. (2005)
Pathogen adaptation under imperfect vaccination: implications for pertussis. Proc Biol Sci
272: 1617-1624
139. Harris K. A., Underwood A., Kenna D. T.,
Brooks A., Kavaliunaite E., et al. (2015)
Whole-genome sequencing and epidemiological analysis do not provide evidence forcross-transmission of mycobacterium abscessus in a cohort of pediatric cystic fibrosis
patients. Clin Infect Dis 60: 1007-1016
140. Tettelin H., Davidson R. M., Agrawal S., Aitken
M. L., Shallom S., et al. (2014) High-level
relatedness among Mycobacterium abscessus
subsp. massiliense strains from widely separated outbreaks. Emerg Infect Dis 20: 364-371
141. Uchiya K., Takahashi H., Nakagawa T., Yagi T.,
Moriyama M., et al. (2015) Characterization of
a novel plasmid, pMAH135, from Mycobacterium avium subsp. hominissuis. PLoS One 10:
e0117797
142. Singh P., Benjak A., Schuenemann V. J.,
Herbig A., Avanzi C., et al. (2015) Insight into
the evolution and origin of leprosy bacilli fromthe genome sequence of Mycobacterium
lepromatosis. Proc Natl Acad Sci U S A 112:4459-4464
143. Ngeow Y. F., Wong Y. L., Tan J. L., Hong K.
W., Ng H. F., et al. (2015) Identification of new
genomospecies in the Mycobacterium terrae
complex. PLoS One 10: e0120789
144. Kumar V. and Robbins S. L. (2007) Robbins
basic pathology. xiv, 946 p.
145. Ablordey A. S., Vandelannoote K., Frimpong I.
A., Ahortor E. K., Amissah N. A., et al. (2015)
Whole genome comparisons suggest random
distribution of Mycobacterium ulceransgenotypes in a Buruli ulcer endemic region of
Ghana. PLoS Negl Trop Dis 9: e0003681
146. Eldholm V., Monteserin J., Rieux A., Lopez B.,
Sobkowiak B., et al. (2015) Four decades oftransmission of a multidrug-resistant Mycobacterium tuberculosis outbreak strain. Nat
Commun 6: 7119
147. Coll F., McNerney R., Preston M. D., Guerra-Assuncao J. A., Warry A., et al. (2015)
Rapid determination of anti-tuberculosis drug
resistance from whole-genome sequences.
Genome Med 7: 51

An overview of recent publications featuring Illumina technology

45

Early efforts to develop TB drugs. Spores bred from the master strain of tuberculosis are
made into a “suspension” with distilled water and then injected into the prepared liquor.

Review
Galagan J. E. (2014) Genomic insights into tuberculosis. Nat Rev Genet 15: 307-320
Lechartier B., Rybniker J., Zumla A. and Cole S. T. (2014) Tuberculosis drug discovery in the post-postgenomic era. EMBO Mol Med 6: 158-168

References
Ali A., Hasan Z., McNerney R., Mallard K., Hill-Cawthorne G., et al. (2015) Whole genome sequencing
based characterization of extensively drug-resistant Mycobacterium tuberculosis isolates from
Pakistan. PLoS One 10: e0117771
The spread of extensively drug-resistant (XDR) M. tuberculosis calls for improved methods to detect thesestrains and its variants in a reliable manner. In this study, WGS was applied to 37 XDR strains from Pakistan
to investigate genes related to drug resistance. A variable number of SNPs and mutations in genes previously
identified as part of the resistance complex were observed, although several additional changes also
took place. The authors estimate that the concordance between phenotypic and genotypic testing using
commercial assays would be variable, which argues for inclusion of expanded targets for drug resistancedetection in M. tuberculosis.
Illumina Technology: HiSeq 2000
Colman R. E., Schupp J. M., Hicks N. D., Smith D. E., Buchhagen J. L., et al. (2015) Detection of LowLevel Mixed-Population Drug Resistance in Mycobacterium tuberculosis Using High Fidelity Amplicon
Sequencing. PLoS One 10: e0126626
Low-levels of drug-resistant (DR) subpopulations in M. tuberculosis infections are often undetected anduntreated, and may lead to DR tuberculosis. The authors developed and tested a method combining “singlemolecule-overlapping reads” (SMOR) analysis with NGS for determination of ultrarare target alleles fromclinical sputum samples. This method enables evaluation of the clinical relevance of previously undetected,
low-level DR subpopulations and may support interventions to suppress them before they become
clinically relevant.
Illumina Technology: MiSeq v3 300 bp PE reads
Curtis J., Luo Y., Zenner H. L., Cuchet-Lourenco D., Wu C., et al. (2015) Susceptibility to tuberculosis
is associated with variants in the ASAP1 gene encoding a regulator of dendritic cell migration. Nat
Genet 47: 523-527
TB is a major health problem in developing countries, and host genetic factors may be important indetermining susceptibility to the disease. Expression arrays analysis of healthy and pulmonary TB cohorts
showed an association between TB and variants in introns of the ASAP1 gene on chromosome 8q24.
Experimentally, depletion of ASAP1 in DCs showed impaired matrix degradation and migration. The ASAP1
protein is involved in actin and membrane remodeling and has been associated with podosomes. Thus, apotential mechanism of predisposition to TB might involve impaired migration of DCs, due to excessive
reduction of ASAP1 expression.
Illumina Technology: Illumina HT-12 expression array

46

Microbes and metagenomics in human healthMerker M., Blin C., Mona S., Duforet-Frebourg N., Lecher S., et al. (2015) Evolutionary history andglobal spread of the Mycobacterium tuberculosis Beijing lineage. Nat Genet 47: 242-249
The authors tracked the evolutionary history and biogeographical structure of M. tuberculosis of the Beijing
lineage by genetic analysis of 4987 isolates from 99 countries and WGS of 110 representative isolates, thelargest data set ever analyzed. They conclude that this lineage originated in the Far East and then irradiatedworldwide in several waves, coinciding with the Industrial Revolution, the First World War, and HIV epidemics.
Two MDR clones spread throughout Asia and Russia concomitantly with the collapse of public health systems
in the former Soviet Union. The study identifies mutations associated with virulence that may have favored
successful expansion across the world.
Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq
Periwal V., Patowary A., Vellarikkal S. K., Gupta A., Singh M., et al. (2015) Comparative whole-genome
analysis of clinical isolates reveals characteristic architecture of Mycobacterium tuberculosis
pangenome. PLoS One 10: e0122979
Comparative genome analysis is a useful technique to determine the genetic diversity of species considered
fairly homogeneous, such as M. tuberculosis. The authors integrated previously obtained genome assemblies
and included newly sequenced Indian M. tuberculosis isolates. They found a “hard-core genome” shared
among all tested isolates, a “soft-core genome” shared by at least 95% of strains, and an accessory genome.
The study suggests that M. tuberculosis has an open pangenome, and that this approach becomes criticalwhen identifying and selecting appropriate targets for isolate typing.
Illumina Technology: Genome AnalyzerIIx
Rock J. M., Lang U. F., Chase M. R., Ford C. B., Gerrick E. R., et al. (2015) DNA replication fidelity in
Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader. Nat Genet 47: 677-681
DNA replication fidelity relies on proofreading activity of the ε-exonuclease of the replisome in E. coli systems.
However, this unit is dispensable in M. tuberculosis. NGS and other techniques were used in Mycobacterium
to show that the PHP domain of the DnaE1 polymerase performs the exonuclease function, and its absence
significantly increases the mutation rate. Phylogenetic analysis showed that the PHP domain–mediatedactivity is conserved and may be the ancestral prokaryotic proofreader. These findings are relevant for thedevelopment of antimicrobials that target replication fidelity.
Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq v2 100 bp PE reads
Venkatasubramanian S., Dhiman R., Paidipally P., Cheekatla S. S., Tripathi D., et al. (2015) A rho GDP
dissociation inhibitor produced by apoptotic T-cells inhibits growth of Mycobacterium tuberculosis.
PLoS Pathog 11: e1004617
The mechanisms underlying latent M. tuberculosis infection remain poorly understood. The authors
identified a subpopulation of CD4 cells (CD4+CD25+) that produced a soluble factor (D4GDI) able to inhibit
the growth of M. tuberculosis in human macrophages. These cells enhanced production of IL-1β, TNF-α,and ROS, increased apoptosis of infected cells, and induced expression of dormancy survival regulators
in M. tuberculosis. Overall, this study provides evidence of enhanced immunity against M. tuberculosis by
CD4+CD25+D4GDI+ cells.
Illumina Technology: HumanHT-12_V4 Expression BeadChip arrays
Wada T., Iwamoto T., Tamaru A., Seto J., Ahiko T., et al. (2015) Clonality and micro-diversity of anationwide spreading genotype of Mycobacterium tuberculosis in Japan. PLoS One 10: e0118495
M. tuberculosis “M strain” has spread recently throughout Japan. The authors performed deep sequencingof 10 M strain isolates to uncover the history of its spread. All the isolates possessed mutations common
to those of referential strains, and accumulated single-nucleotide variants (SNVs) with evidence of highclonality of these isolates. Further analysis revealed distribution into 3 subclonal groups with a bias toward
geographical origins. These efforts inform public health actions against TB by providing efficient strategies forbig data analysis.
Illumina Technology: Genome AnalyzerIIx75 bp PE reads
Ablordey A. S., Vandelannoote K., Frimpong I. A., Ahortor E. K., Amissah N. A., et al. (2015) Whole genome
comparisons suggest random distribution of Mycobacterium ulcerans genotypes in a Buruli ulcer endemic
region of Ghana. PLoS Negl Trop Dis 9: e0003681
Harris K. A., Underwood A., Kenna D. T., Brooks A., Kavaliunaite E., et al. (2015) Whole-genome sequencing
and epidemiological analysis do not provide evidence for cross-transmission of mycobacterium abscessus ina cohort of pediatric cystic fibrosis patients. Clin Infect Dis 60: 1007-1016
Ngeow Y. F., Wong Y. L., Tan J. L., Hong K. W., Ng H. F., et al. (2015) Identification of new genomospecies in
the Mycobacterium terrae complex. PLoS One 10: e0120789

An overview of recent publications featuring Illumina technology

47

Singh P., Benjak A., Schuenemann V. J., Herbig A., Avanzi C., et al. (2015) Insight into the evolution andorigin of leprosy bacilli from the genome sequence of Mycobacterium lepromatosis. Proc Natl Acad Sci U S A
112: 4459-4464
Anderson S. T., Kaforou M., Brent A. J., Wright V. J., Banwell C. M., et al. (2014) Diagnosis ofchildhood tuberculosis and host RNA expression in Africa. N Engl J Med 370: 1712-1723
There is a need for improved diagnostic tests for TB in children. This study defined the transcriptionalsignatures of host blood samples from children in South Africa, Malawi, and Kenya, distributed according toculture status. The authors identified a 51-transcript signature distinguishing TB from other diseases in South
African and Malawian children. These results were validated in the Kenyan cohort to determine the sensitivity
and specificity against culture-confirmed and culture-negative diagnoses. RNA expression signatures helped
distinguish TB from other diseases in African children with and without HIV infection.
Illumina Technology: HumanHT-12_V4 Expression BeadChip arrays
Li D., Gao G., Li Z., Sun W., Li X., et al. (2014) Profiling the T-cell receptor repertoire of patient withpleural tuberculosis by high-throughput sequencing. Immunol Lett 162: 170-180
Pleural effusion is a common manifestation of pulmonary TB. However, the T-cell receptor (TCR) role andrepertories of pleural effusion mononuclear cells (PEMCs) have not been elucidated. In this study, NGS wasapplied to PEMCs and peripheral blood mononuclear cells (PBMCs) collected from a single patient who
had developed pleural effusion. The authors describe the genetic profile of TCR β-chain receptors, including
different CDR3 clonotypes and patterns, in both PEMCs and PBMCs.
Illumina Technology: Genome AnalyzerIIx
Lin P. L., Ford C. B., Coleman M. T., Myers A. J., Gawande R., et al. (2014) Sterilization of granulomas
is common in active and latent tuberculosis despite within-host variability in bacterial killing. Nat Med
20: 75-79
Some individuals remain latently infected with TB, although the reasons for this inactive infection are still
obscure. A similar pattern is found in macaques experimentally infected with individually tagged bacteria. This
study showed how each lung lesion is likely founded by a single cell. However, the fate of each lesion was
different in each individual, i.e., some active lesions were sterilized by the host whereas others progressed.
This study suggests that critical responses at the lesion level determine the outcome of infection.
Illumina Technology: Genome AnalyzerIIx

Escherichia coli
Commonly found in the environment, food products, and intestines of people andanimals, most strains of E. coli are commensals. However, some strains can bepathogens that enjoy high transmissibility and often cause foodborne outbreaks.
The pathotype Shiga toxin-producing E. coli (STEC) is perhaps the most common
example of this group, although 5 other pathotypes are recognized. Rapid
identification is essential to track and control foodborne outbreaks to their sources.148

ReviewFranz E., Delaquis P., Morabito S., Beutin L., Gobius K., et al. (2014) Exploiting the explosion of information
associated with whole genome sequencing to tackle Shiga toxin-producing Escherichia coli (STEC) in global
food production systems. Int J Food Microbiol 187: 57-72

References
Griffing S. M., MacCannell D. R., Schmidtke A. J., Freeman M. M., Hyytia-Trees E., et al. (2015)
Canonical Single Nucleotide Polymorphisms (SNPs) for High-Resolution Subtyping of Shiga-Toxin
Producing Escherichia coli (STEC) O157:H7. PLoS One 10: e0131967
The authors aimed to develop a SNP panel for subtyping of STEC O157:H7 that would be consistent withother available discrimination methods. An initial screening of a large SNP panel failed to resolve somebacterial clusters accurately, forcing a second screening using comparative DNA resequencing from theclusters with poor resolution. This method resulted in increase of SNP panel size, with 63 SNPs previously
identified and 438 occurring across multiple clusters. The authors conclude that linkage disequilibrium limits
the number of informative SNPs and that these SNPs are inefficient for effective subtyping.
Illumina Technology: TruSeq DNA Library Prep Kit, MiSeq

48

Microbes and metagenomics in human health148. Lambert D., Carrillo C. D., Koziol A. G., Manninger P. and Blais B. W. (2015) GeneSippr:
a rapid whole-genome approach for theidentification and characterization of foodborne
pathogens such as priority Shiga toxigenic
Escherichia coli. PLoS One 10: e0122928

Lambert D., Carrillo C. D., Koziol A. G., Manninger P. and Blais B. W. (2015) GeneSippr: a rapid wholegenome approach for the identification and characterization of foodborne pathogens such as priority
Shiga toxigenic Escherichia coli. PLoS One 10: e0122928
The authors describe a WGS approach that enables same-day identification of colony isolates recoveredfrom investigative food samples. The method used 21-nt reads and 4-fold coverage followed by custom
data analysis software, called GeneSippr. Pathogens were correctly identified in all isolates tested, and single
colonies were identified within 9 hours.
Illumina Technology: Nextera XT DNA Library Prep Kit, Nextera XT Index Kit, MiSeq v2

149. Hartman B. J. and Tomasz A. (1984) Low-affinity penicillin-binding protein associated withbeta-lactam resistance in Staphylococcus
aureus. J Bacteriol 158: 513-516
150. Pinho M. G., de Lencastre H. and Tomasz A.
(2001) An acquired and a native penicillin-binding protein cooperate in building the cell wall of
drug-resistant staphylococci. Proc Natl Acad
Sci U S A 98: 10886-10891

Zhang Y., Rowehl L., Krumsiek J. M., Orner E. P., Shaikh N., et al. (2015) Identification of Candidate
Adherent-Invasive E. coli Signature Transcripts by Genomic/Transcriptomic Analysis. PLoS One 10:
e0130902
Adherent-invasive E. coli (AIEC) is frequently found in mucosal lesions of patients with Crohn’s disease. RNASeq was used to identify differentially expressed genes in an AIEC strain relative to a noninvasive E. coli (NIEC)
strain that may account for difference in phenotypes. The results showed that 224 and 241 genes wereupregulated and downregulated in AIEC, respectively. The authors describe the functions of these enrichedgenes and pathways, as well as genes and CRISPR-Cas loci exclusive to AIEC strains. These findings wereconfirmed in IBD and non-IBD fecal RNA samples that demonstrated enrichment of CRISPR-Cas in IBD. Thisapproach is useful to identify candidate AIEC signature transcripts.
Illumina Technology: MiSeq 150 bp SE reads
Salipante S. J., Roach D. J., Kitzman J. O., Snyder M. W., Stackhouse B., et al. (2015) Large-scale genomic
sequencing of extraintestinal pathogenic Escherichia coli strains. Genome Res 25: 119-128
von Mentzer A., Connor T. R., Wieler L. H., Semmler T., Iguchi A., et al. (2014) Identification ofenterotoxigenic Escherichia coli (ETEC) clades with long-term global distribution. Nat Genet 46: 1321-1326
Enterotoxigenic E. coli (ETEC) is an important cause of diarrhea by producing heat-stable and/or heat-labile
toxins, along with several colonization factors that are encoded in plasmids. The authors performed WGS ofa collection of globally distributed ETEC strains that carry distinct colonization factors and enterotoxin profiles.
Challenging previous notions, these recent lineages might contain chromosome and plasmids combinations
that optimize fitness and transmissibility, with implications for tracking ETEC outbreaks.
Illumina Technology: HiSeq 2000 100 bp PE reads
Bielaszewska M., Schiller R., Lammers L., Bauwens A., Fruth A., et al. (2014) Heteropathogenic virulence andphylogeny reveal phased pathogenic metamorphosis in Escherichia coli O2:H6. EMBO Mol Med 6: 347-357
Petty N. K., Ben Zakour N. L., Stanton-Cook M., Skippington E., Totsika M., et al. (2014) Global
dissemination of a multidrug resistant Escherichia coli clone. Proc Natl Acad Sci U S A 111: 5694-5699
Xu H., Zhu X., Hu Y., Li Z., Zhang X., et al. (2014) DNA methylome in spleen of avian pathogenic Escherichia
coli-challenged broilers and integration with mRNA expression. Sci Rep 4: 4299

Staphylococcus aureus
This Gram-positive pathogen has gained notoriety because of its antimicrobial
resistance capabilities. Life-threatening infections caused by this agent in hospitalsettings are difficult to control. S. aureus encodes 4 (methicillin-susceptible, MSSA)
or 5 (methicillin-resistant, MRSA) penicillin-binding proteins (PBP) involved in cellwall peptidoglycan (PG) synthesis. The extra PBP2A in MRSA is a determinant inresistance against β-lactams.149, 150

References
Reed P., Atilano M. L., Alves R., Hoiczyk E., Sher X., et al. (2015) Staphylococcus aureus Survives witha Minimal Peptidoglycan Synthesis Machine but Sacrifices Virulence and Antibiotic Resistance. PLoS
Pathog 11: e1004891
Peptidoglycan is the main component of the cell wall, and a machinery of 9 proteins is required for its
synthesis in S. aureus. This study examined the genomic and transcriptomic effects of deleting 7 of the
9 proteins of this machinery. The authors identified PBP1 and PBP2 as the minimal machinery requiredfor PG synthesis in vitro. However, the rest of the complex seems to be required under more challenging
environments, as the minimal machinery is susceptible to a variety of conditions. Thus, the complex could
have a role in pathogenesis and antibiotic resistance.
Illumina Technology: MiSeq, HiSeq

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Tong S. Y., Holden M. T., Nickerson E. K., Cooper B. S., Koser C. U., et al. (2015) Genome sequencing
defines phylogeny and spread of methicillin-resistant Staphylococcus aureus in a high transmission
setting. Genome Res 25: 111-118
WGS was used to establish the flux and genetic diversity of MRSA in 2 intensive-care units wheretransmission was common. A total of 79 isolates were sequenced, belonging to distinct ST 239 clades thatcarried different plasmids encoding determinants for antibiotic resistance. Inter- and intra-ward transmission
events and multiple colonizations by more than 1 clade were identified. WGS provides insights into thetransmission and spread of MRSA, and this approach can be applied in different settings.
Illumina Technology: Genome AnalyzerIIx 100 bp PE reads, HiSeq 75 bp PE reads
Brady R. A., Bruno V. M. and Burns D. L. (2015) RNA-Seq Analysis of the Host Response to Staphylococcus
aureus Skin and Soft Tissue Infection in a Mouse Model. PLoS One 10: e0124877
Ellington M. J., Reuter S., Harris S. R., Holden M. T., Cartwright E. J., et al. (2015) Emergent andevolving antimicrobial resistance cassettes in community-associated fusidic acid and meticillin-resistant
Staphylococcus aureus. Int J Antimicrob Agents 45: 477-484
Everitt R. G., Didelot X., Batty E. M., Miller R. R., Knox K., et al. (2014) Mobile elements drive
recombination hotspots in the core genome of Staphylococcus aureus. Nat Commun 5: 3956
Genetic exchange in the core genomes of clonal bacteria like S. aureus is not fully understood, despite
increasing evidence about mobile elements. The authors performed WGS of MRSA and MSSA historical,
international, human, and animal strains to gain insight into core genomes. They found recombination
hotspots in close proximity to known mobile elements. This type of mobile element–driven coregenome transfer broadens our understanding of clonal bacteria, with a possible impact on phenotypegenotype mapping.
Illumina Technology: Genome AnalyzerIIx HiSeq 2000 100 bp PE reads

Salmonella
Salmonella enterica serovar Enteritidis and serovar Typhimurium are important leading
causes of foodborne gastroenteritis in humans, although many other serovars alsopose health risks for humans and animals. Serovar Enteritidis is prevalent in thepoultry industry. It is particularly efficient at replicating within chicken eggs, making
identification and origin tracing crucial for surveillance and to control foodborneassociated outbreaks. Salmonella is a genetically homogeneous bacterium, andgenome sequencing provides accurate differentiation among strains.151

Food-borne outbreaks of Salmonella Enteritidis are often associated with chicken eggs and poultry products.
NGS supports public health surveillance and allows differentiation among genetically homogeneous strains.

Review
Park S. H., Aydin M., Khatiwara A., Dolan M. C., Gilmore D. F., et al. (2014) Current and emerging
technologies for rapid detection and characterization of Salmonella in poultry and poultry products. Food
Microbiol 38: 250-262

50

Microbes and metagenomics in human health151. den Bakker H. C., Allard M. W., Bopp D.,
Brown E. W., Fontana J., et al. (2014) Rapid
whole-genome sequencing for surveillance of
Salmonella enterica serovar enteritidis. Emerg
Infect Dis 20: 1306-1314

References
Inns T., Lane C., Peters T., Dallman T., Chatt C., et al. (2015) A multi-country Salmonella Enteritidis
phage type 14b outbreak associated with eggs from a German producer: ‘near real-time’ applicationof whole genome sequencing and food chain investigations, United Kingdom, May to September
2014. Euro Surveill 20:
The authors report an outbreak of Salmonella Enteritidis phage type 14b (PT14b) in the UK. The outbreak
was related to a specific, multiple-locus variable number tandem repeat analysis (MLVA) pattern that occurred
simultaneously in other countries in the European Union. Epidemiological data, MLVA patterns, and WGS
demonstrated that the outbreaks were linked to chicken eggs that originated from a German company. In thiscase, the use of genome sequence information was useful to trace back foodborne outbreaks to their source
in a timely manner to implement control measures.
Illumina Technology: HiSeq 2500

152. de Castro A. P., Fernandes Gda R. and Franco
O. L. (2014) Insights into novel antimicrobial
compounds and antibiotic resistance genes
from soil metagenomes. Front Microbiol 5: 489
153. Bhullar K., Waglechner N., Pawlowski A.,
Koteva K., Banks E. D., et al. (2012) Antibiotic
resistance is prevalent in an isolated cave
microbiome. PLoS One 7: e34953
154. Li B., Yang Y., Ma L., Ju F., Guo F., et al. (2015)
Metagenomic and network analysis reveal
wide distribution and co-occurrence of environmental antibiotic resistance genes. ISME J

Feasey N. A., Gaskell K., Wong V., Msefula C., Selemani G., et al. (2015) Rapid emergence of multidrug
resistant, H58-lineage Salmonella typhi in Blantyre, Malawi. PLoS Negl Trop Dis 9: e0003748
Hamidian M., Holt K. E. and Hall R. M. (2015) The complete sequence of Salmonella genomic island SGI1-K.
J Antimicrob Chemother 70: 305-306
Schreiber F., Kay S., Frankel G., Clare S., Goulding D., et al. (2015) The Hd, Hj, and Hz66 flagella variants ofSalmonella enterica serovar Typhi modify host responses and cellular interactions. Sci Rep 5: 7947
Deng X., Desai P. T., den Bakker H. C., Mikoleit M., Tolar B., et al. (2014) Genomic epidemiology of
Salmonella enterica serotype Enteritidis based on population structure of prevalent lineages. Emerg
Infect Dis 20: 1481-1489
Salmonella enterica is a leading cause of foodborne gastroenteritis worldwide, and surveillance is critical to
implement control measures during outbreaks. However, it is genetically homogeneous and, thus, subtyping
is challenging. The authors used WGS to characterize 125 S. enterica Enteritidis and 3 S. enterica Nitra
strains, together with SNP analysis to identify 4887 loci that distinguished all isolates from each other. Thisapproach identified 5 major genetic lineages, possibly related to geographic and epidemiologic patterns that
may have spawned during the 17th–18th centuries and diversified during the 1920s and 1950s.
Illumina Technology: HiSeq 2000
Leekitcharoenphon P., Nielsen E. M., Kaas R. S., Lund O. and Aarestrup F. M. (2014) Evaluation ofwhole genome sequencing for outbreak detection of Salmonella enterica. PLoS One 9: e87991
Reliable subtyping of Salmonella enterica is essential for surveillance and foodborne outbreak responses.
Here, 34 S. Typhimurium, 8 S. Enteritidis, and 5 S. Derby isolates were sequenced to evaluate WGS as
an epidemiological typing tool. Four different phylogenetic analyses (pan-genome, K-mer, SNP, nucleotide
difference trees) were applied to the S. Typhimurium data and compared to data from pulse-field gelelectrophoresis. The results showed that WGS achieved greater performance than traditional typing methods.
SNP and nucleotide-difference analytic approaches for WGS data were better methods for epidemiologicaltyping compared to other approaches. However, combination of epidemiological data and WGS is stillnecessary to relate strains to outbreaks.
Illumina Technology: Genome AnalyzerIIx
Maudet C., Mano M., Sunkavalli U., Sharan M., Giacca M., et al. (2014) Functional high-throughput screening
identifies the miR-15 microRNA family as cellular restriction factors for Salmonella infection. Nat Commun 5: 4718

Antimicrobial ResistanceResistance to antimicrobial compounds is an urgent public health concern,with increased patient mortality and health care costs.152 Multiple geneticallyencoded mechanisms play a role in resistance and can be transferred via mobile
elements between bacteria, leading to the spread of resistance to single ormultiple antimicrobials.
Antibiotic-resistance genes are ancient and ubiquitous,153 but interaction with people,
animals, and the environment may have facilitated their selection and spread.154
Agricultural practices also impact the spread of antibiotic-resistance genes in soil

An overview of recent publications featuring Illumina technology

51and food animals.155 Aquaculture practices use extreme amounts of antibiotics incertain salmon-producing countries,156 whereas global consumption of antimicrobials
is expected to increase by 67% in livestock animals by 2030.157 Culture-independent
in silico antimicrobial susceptibility testing to support surveillance efforts and provide
tracking of drug resistant pathogens158 can be achieved by using information
provided by genomic analyses.
Genomic approaches can help reveal many of these genes, identify new ones, unveil
in situ resistance mechanisms, and potentially identify genes encoding antimicrobial
compounds. Soil is probably the richest source of antimicrobials and antimicrobialresistance genes,

159, 160

with many compounds still locked in the unculturable

bacterial world.161

155. Udikovic-Kolic N., Wichmann F., Broderick
N. A. and Handelsman J. (2014) Bloom ofresident antibiotic-resistant bacteria in soil
following manure fertilization. Proc Natl Acad
Sci U S A 111: 15202-15207
156. Henriksson P. J., Troell M. and Rico A. (2015)
Antimicrobial use in aquaculture: Some
complementing facts. Proc Natl Acad Sci U S
A 112: E3317
157. Van Boeckel T. P., Brower C., Gilbert M., Grenfell B. T., Levin S. A., et al. (2015) Global trends
in antimicrobial use in food animals. Proc Natl
Acad Sci U S A 112: 5649-5654
158. Fricke W. F. and Rasko D. A. (2014) Bacterial
genome sequencing in the clinic: bioinformaticchallenges and solutions. Nat Rev Genet 15:
49-55
159. de Castro A. P., Fernandes Gda R. and Franco
O. L. (2014) Insights into novel antimicrobial
compounds and antibiotic resistance genes
from soil metagenomes. Front Microbiol 5: 489
160. Nesme J., Cecillon S., Delmont T. O., Monier
J. M., Vogel T. M., et al. (2014) Large-scale
metagenomic-based study of antibiotic
resistance in the environment. Curr Biol 24:
1096-1100
161. Ling L. L., Schneider T., Peoples A. J.,
Spoering A. L., Engels I., et al. (2015) A new
antibiotic kills pathogens without detectable
resistance. Nature 517: 455-459

Genomic information of antimicrobial resistance in health care systems can inform implementation ofcontrol measures and evaluate their effectiveness.

Reviews
Hughes D. and Andersson D. I. (2015) Evolutionary consequences of drug resistance: shared principles
across diverse targets and organisms. Nat Rev Genet 16: 459-471
Mather A. E. (2015) There’s no place like home. Nat Rev Microbiol 13: 331
Nesme J. and Simonet P. (2015) The soil resistome: a critical review on antibiotic resistance origins, ecology
and dissemination potential in telluric bacteria. Environ Microbiol 17: 913-930
van Schaik W. (2015) The human gut resistome. Philos Trans R Soc Lond B Biol Sci 370: 20140087doi:
10.1098/rstb.2014.0087
Woolhouse M., Ward M., van Bunnik B. and Farrar J. (2015) Antimicrobial resistance in humans, livestockand the wider environment. Philos Trans R Soc Lond B Biol Sci 370: 20140083
de Castro A. P., Fernandes Gda R. and Franco O. L. (2014) Insights into novel antimicrobial compounds andantibiotic resistance genes from soil metagenomes. Front Microbiol 5: 489
Koser C. U., Ellington M. J. and Peacock S. J. (2014) Whole-genome sequencing to control antimicrobial
resistance. Trends Genet 30: 401-407
Mullany P. (2014) Functional metagenomics for the investigation of antibiotic resistance. Virulence 5: 443-447

References
Hsu L. Y., Harris S. R., Chlebowicz M. A., Lindsay J. A., Koh T. H., et al. (2015) Evolutionary dynamics
of methicillin-resistant Staphylococcus aureus within a healthcare system. Genome Biol 16: 81
MRSA ST239 has seen some replacement with ST22 clones that are more susceptible to antimicrobial
treatment over the past few years. To understand the historical and evolutionary features, the authors
performed WGS and phylogenetic analysis of ST239 and ST22 in Singapore hospitals. They found that52

Microbes and metagenomics in human healthST239 was introduced from adjacent regions and then spread in the health care system. ST22 wasintroduced around the year 2000, supplanting ST239. The genetic diversity of ST239 appeared to decrease
as ST22 replaced it. However, diversity increased once more, around the year 2007 and later. The study
shows the importance of hospital practices and competition between clones to drive pathogen evolution.
Illumina Technology: Genome AnalyzerIIx, HiSeq
Pecora N. D., Li N., Allard M., Li C., Albano E., et al. (2015) Genomically Informed Surveillance for
Carbapenem-Resistant Enterobacteriaceae in a Health Care System. MBio 6: e01030
WGS allows refined tracking of chromosomal traits and associated mobile genetic elements that harbor
resistance genes. This information can be used to alert hospitals to prevent the spread of infections.
Isolates of Klebsiella pneumoniae and Enterobacter cloacae with resistance to carbapenem antibiotics
were sequenced. This study identified multiple carbapenemase genes residing in distinct mobile elementsthat provided strain heterogeneity. The authors were able to draft a timeline of carbapenemase entry andmovement in a hospital setting. This type of study supports infection control resources and the developmentof resistance gene repositories at an institutional level.

162. Sutton P. (2015) At last, vaccine-induced protection against Helicobacter pylori. Lancet
163. Moustafa D. A., Scarff J. M., Garcia P. P.,
Cassidy S. K., DiGiandomenico A., et al.
(2015) Recombinant Salmonella Expressing
Burkholderia mallei LPS O Antigen Provides
Protection in a Murine Model of Melioidosis
and Glanders. PLoS One 10: e0132032.
164. Xu X., Hegazy W. A., Guo L., Gao X., Courtney
A. N., et al. (2014) Effective cancer vaccine
platform based on attenuated salmonella anda type III secretion system. Cancer Res 74:
6260-6270
165. Saxena M., Van T. T., Baird F. J., Coloe P. J.
and Smooker P. M. (2013) Pre-existing immunity against vaccine vectors--friend or foe?
Microbiology 159: 1-11

Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq v1 150 bp PE reads, MiSeq v3 300 bp PE reads

Bacterial Vaccines
Vaccines against C. tetani are highly effective, and their use is routine. On the otherend of the spectrum, for microorganisms such as H. pylori, vaccine development
is still elusive, despite the potential utility in prevention of ulcer disease and gastricadenocarcinoma.162
Long-dreaded microbes now have a chance to contribute to immunotherapy as
antigen-expressing platforms. Attenuated Salmonella Typhimurium expressing
B. mallei LPS O antigen has been successful at protecting mice against lethal
challenges.163 Similarly, Salmonella T3SS has been used to deliver tumoral antigens,enhance antitumor immunity, and induce tumor regression in the colon carcinoma
mouse model.164 Some pre-existing immunity against these vectors could actuallyenhance immunogenicity.165

References
Croucher N. J., Chewapreecha C., Hanage W. P., Harris S. R., McGee L., et al. (2014) Evidence forsoft selective sweeps in the evolution of pneumococcal multidrug resistance and vaccine escape.
Genome Biol Evol 6: 1589-1602
S. pneumoniae PMEM14 serotype 19F is targeted by the PCV7 heptavalent vaccine. Unfortunately, there
is an emergence of vaccine-escape variants. The authors performed WGS of 173 PMEM14 or relatives
to characterize its evolution. PMEM14 is a single lineage with 4 serotype switches to 19A, all with different
resistance to β-lactams through homologous recombinations that also occur in absence of vaccination.
These findings suggest that pneumococcal genotypes generate pools of standing variation that result in theemergence of multiple mutants, in parallel, upon changes in selection pressure.
Illumina Technology: Genome AnalyzerIIx, HiSeq
Cohen-Gihon I., Israeli O., Beth-Din A., Levy H., Cohen O., et al. (2014) Whole-Genome Sequencingof the Nonproteolytic Bacillus anthracis V770-NP1-R Strain Reveals Multiple Mutations in Peptidase
Loci. Genome Announc 2: e00075-14.
Anthrax is a severe disease caused by inhalation/ingestion of, or contact of skin abrasions with, B. anthracis
spores. The spores produce vegetative bacilli, resulting in massive bacteremia and toxemia. The authors
generated a draft whole-genome sequence of the nonproteolytic Bacillus anthracis V770-NP1-R strain.
Compared to those of other B. anthracis strains, the genome exhibits unique mutations in multiple targets
potentially affecting proteolytic functions. One of these mutations is a deletion that disrupts the NprR quorumsensing regulator of the NprA protease.
Illumina Technology: Genome AnalyzerIIx

An overview of recent publications featuring Illumina technology

53

MICROBIAL POPULATIONS
To determine the identity and abundance of microbes in a defined sample, there
are 2 sequencing approaches: amplicon sequencing and whole-genome shotgun
sequencing (Table 3). Amplicon sequencing relies on PCR amplification andsequencing of the 16S rRNA gene as a single informative marker.166 Whole-genome
shotgun sequencing refers to random shearing of whole extracted DNA and thensequencing the small DNA fragments. Both approaches offer distinct advantages
and trade-offs. Selecting an appropriate approach depends on the type of
information required.

167. Diaz P. I., Dupuy A. K., Abusleme L., Reese B.,
Obergfell C., et al. (2012) Using high throughput sequencing to explore the biodiversity inoral bacterial communities. Mol Oral Microbiol
27: 182-201
168. Barone R., De Santi C., Palma Esposito F.,
Tedesco P., Galati F., et al. (2014) Marine
metagenomics, a valuable tool for enzymes
and bioactive compounds discovery. Frontiersin Marine Science 1:

Reviews
Gasc C., Ribiere C., Parisot N., Beugnot R., Defois C., et al. (2015) Capturing prokaryotic dark mattergenomes. Res Microbiol
Reuter J. A., Spacek D. V. and Snyder M. P. (2015) High-throughput sequencing technologies. Mol Cell 58:
586-597

169. Solli L., Havelsrud O. E., Horn S. J. and Rike
A. G. (2014) A metagenomic study of the
microbial communities in four parallel biogas
reactors. Biotechnol Biofuels 7: 146

McAdam P. R., Richardson E. J. and Fitzgerald J. R. (2014) High-throughput sequencing for the study ofbacterial pathogen biology. Curr Opin Microbiol 19: 106-113

170. Olson N. D., Lund S. P., Zook J. M.,
Rojas-Cornejo F., Beck B., et al. (2015) International interlaboratory study comparing single
organism 16S rRNA gene sequencing data:
Beyond consensus sequence comparisons.
Biomolecular Detection and Quantification 3:
17-24

Table 3: Amplicon and Whole-Genome Shotgun Sequencing.

171. Handelsman J. (2004) Metagenomics: application of genomics to uncultured microorganisms. Microbiol Mol Biol Rev 68: 669-685

Feature54

166. Lane D. J., Pace B., Olsen G. J., Stahl D. A.,
Sogin M. L., et al. (1985) Rapid determinationof 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A 82:
6955-6959

16s rRNA(Amplicon
Sequencing)167

Whole-Genome Shotgun Sequencing
168, 169

Type ofinformation
produced

Taxonomic
composition andcommunity structure
as operationaltaxonomic units170

Potentially functional characterizationof the whole community; draft genome
reconstruction of individual community
members

Application

Monitorpopulations171

Detect new members, new genes, resolve
complex populations

Ability to detectrare members of
the community

Highly sensitive
to single target
representing 80% of
total bacterial RNA

Requires deeper sequencing to achieve thesame level of sensitivity

Biases

Probe and
amplification bias;
unable to represent
the whole genomic
content172

Sequence-content bias

Gene inventory

Functionality is largely
unknown

Extensive inventories and partial genomes;
discover new genes and biological pathways

Microbes and metagenomics in human health172. Poretsky R., Rodriguez R. L., Luo C.,
Tsementzi D. and Konstantinidis K. T. (2014)
Strengths and limitations of 16S rRNA gene
amplicon sequencing in revealing temporalmicrobial community dynamics. PLoS One 9:
e93827

Amplicon Sequencing: 16S Ribosomal RNA
The characterization of a single genetic marker, such as the 16S rRNA gene, hasbeen extensively used for assessing the diversity of microbial populations forphylogenetic and taxonomic studies. The 16S rRNA gene exists in all bacteria and iscomposed of interspersed variable regions flanked by relatively conserved regions.173
Probes hybridize to the conserved regions for PCR amplification and sequencing of
the variable regions, which cluster into operational taxonomic units (OTUs) according
to degree of similarity.174 This classical approach has yielded comprehensive
databases for comparison of sequences in an ecosystem and evolutionary analyses
applicable to large projects, such as the Human Microbiome Project.175
As a single marker, 16S rRNA sequencing is able to capture broad changes
in bacterial community diversity, an attractive feature due to the lower cost of
sequencing only a small part of the genome. However, amplicon sequencing does
not provide information about the potential genomic functions of the community, nordoes it detect viruses.

Deep Sequencing to Characterize Complex Populations
Deep sequencing refers to the resequencing of a genomic region up to thousandsof times. This method provides improved representation of all the members of thecommunity, including species that are present in low abundance. In nature, bacteria

173. Vinje H., Almoy T., Liland K. H. and Snipen L.
(2014) A systematic search for discriminatingsites in the 16S ribosomal RNA gene. Microb
Inform Exp 4: 2
174. Schmidt T. S., Matias Rodrigues J. F. andvon Mering C. (2014) Ecological consistencyof SSU rRNA-based operational taxonomic
units at a global scale. PLoS Comput Biol 10:
e1003594
175. McDonald D., Price M. N., Goodrich J.,
Nawrocki E. P., DeSantis T. Z., et al. (2012) An
improved Greengenes taxonomy with explicit
ranks for ecological and evolutionary analyses
of bacteria and archaea. ISME J 6: 610-618
176. Chiarello M., Villeger S., Bouvier C., Bettarel
Y. and Bouvier T. (2015) High diversity ofskin-associated bacterial communities of marine fishes is promoted by their high variability
among body parts, individuals and species.
FEMS Microbiol Ecol 91:
177. Neuenschwander S. M., Pernthaler J., PoschT. and Salcher M. M. (2015) Seasonal growthpotential of rare lake water bacteria suggest
their disproportional contribution to carbon
fluxes. Environ Microbiol 17: 781-795
178. Shade A., Jones S. E., Caporaso J. G., Handelsman J., Knight R., et al. (2014) Conditionally rare taxa disproportionately contribute totemporal changes in microbial diversity. MBio
5: e01371-01314

live in highly diverse populations that are able to change over time and space.
Changes can also occur within and among individuals from the same or different
species.176 Within a community, some microorganisms exist in low abundance,
but they may have significant effects in the community and health status. Thisphenomenon usually becomes apparent if their abundance or functions adjust tochanges in the ecosystem.177, 178 The discrimination power of deep sequencing
is critical to characterize heterogeneous communities fully and to provide betterinterpretation of subtle microbiological changes.

Abundant (Green) and poorly represented (Orange) microbial populations sampled at different depths.
Deep sequencing (Depth B) captures fluctuations in abundance in both types of populations. These changes
are missed when abundance drops below the detection limit in Depth A.

An overview of recent publications featuring Illumina technology

55

The discovery of most high taxa from environmental surveys might be a reality by the end of thisdecade.179 Phylogenetic diversity increases with read depth, and the optimal read depth is when diversity
no longer increases.

Amplification Bias
The determination of microbial diversity is hampered due to PCR amplification andcloning-associated biases. Accuracy depends on primer choice (Table 4).

Table 4: Amplification Bias.

FactorImpactPrimer choice

Coverage
Primer-template binding affinity
Spurious products
Differential annealing capabilities and amplification
efficiency180

Lack of consensus foruniversal primers

Prevents comparison between studies and comprehensive
coverage of bacterial diversity181

Template concentration

Biases the determination of composition and theabundance of microbial communities182

Cloning Bias
Metagenomic clone libraries can be created by cloning large genomic fragments
into cosmids or fosmids that are propagated in well-characterized host vectors, likeE. coli.183 These libraries often exhibit GC biases, poor transcriptional activity of thecloned DNA,184 and occasional toxicity from expressed genes.185 Long-term storage
favors overgrowth of some clones at the expense of others, skewing the overall
library diversity.186

Intragenomic Heterogeneity
Variable copy numbers, and the diversity of 16S rRNA sequences across different
bacterial taxa and within species, are common. Bacteria contain an average of 4
copies of the 16S rRNA gene per genome, reaching a maximum of 15 copies insome Fusobacteria, with sequence variability increasing with copy number.187 Dueto the variability of 16S rRNA copy numbers, amplicon sequencing is limited in its

56

Microbes and metagenomics in human health179. Yarza P., Yilmaz P., Pruesse E., Glockner F. O.,
Ludwig W., et al. (2014) Uniting the classification of cultured and uncultured bacteria andarchaea using 16S rRNA gene sequences. Nat
Rev Microbiol 12: 635-645
180. Ibarbalz F. M., Perez M. V., Figuerola E. L.
and Erijman L. (2014) The bias associatedwith amplicon sequencing does not affect thequantitative assessment of bacterial community dynamics. PLoS One 9: e99722
181. Takahashi S., Tomita J., Nishioka K., Hisada
T. and Nishijima M. (2014) Development ofa prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using
next-generation sequencing. PLoS One 9:e105592
182. Chandler D. P., Fredrickson J. K. and Brockman F. J. (1997) Effect of PCR template concentration on the composition and distributionof total community 16S rDNA clone libraries.
Mol Ecol 6: 475-482
183. Cretoiu M. S., Berini F., Kielak A. M., Marinelli
F. and van Elsas J. D. (2015) A novel salt-tolerant chitobiosidase discovered by genetic
screening of a metagenomic library derived
from chitin-amended agricultural soil. Appl
Microbiol Biotechnol 99: 8199-8215
184. Lam K. N. and Charles T. C. (2015) Strong
spurious transcription likely contributes to
DNA insert bias in typical metagenomic clone
libraries. Microbiome 3: 22
185. Kimelman A., Levy A., Sberro H., Kidron S.,
Leavitt A., et al. (2012) A vast collection of
microbial genes that are toxic to bacteria.
Genome Res 22: 802-809
186. Chandler D. P., Brockman F. J. and Fredrickson J. K. (1997) Use of 16S rDNA clone
libraries to study changes in a microbialcommunity resulting from ex situ perturbationof a subsurface sediment. FEMS Microbiology
Reviews 20: 217-230
187. Vetrovsky T. and Baldrian P. (2013) The
variability of the 16S rRNA gene in bacterial
genomes and its consequences for bacterial
community analyses. PLoS One 8: e57923
188. Rajendhran J. and Gunasekaran P. (2011)
Microbial phylogeny and diversity: small
subunit ribosomal RNA sequence analysis andbeyond. Microbiol Res 166: 99-110

ability to estimate bacterial community richness and abundance accurately. More
importantly, amplicon sequencing cannot provide information on the genetic potentialof the community.188

Horizontal Gene Transfer and Mosaicism
With 16S rRNA amplicon sequencing, it is not possible to assess horizontal
gene transfer or genomic rearrangements outside or within the 16S rRNA genes.
Interspecies exchange of complete 16S rRNA genes, and mosaicism within
the operon by horizontal gene transfer, are tolerated in natural and experimental
conditions due to conservation of secondary and tertiary ribosomal structures thatprovide functional plasticity.189 The identification of species by amplicon sequencing
may lead to incorrect taxa assignment and abundance biases.190

Metagenome Sequencing: Whole-Genome
Shotgun Metagenomics
Metagenome sequencing, or whole-genome shotgun sequencing (Table 5), is themassively parallel sequencing of DNA fragments obtained from the complete gene
repertoire in a microbial population. A major advantage of shotgun WGS is theability to get an accurate phylogenetic tree, as well as the functional profiling of thecommunity, in a single experiment. In functional profiling, the biological pathways
represented in the community are reconstructed to determine in which primaryactivities the community is engaged.
CRISPR/Cas elements are critical in the bacteria’s defense against foreign DNA.
The use of CRISPR/Cas elements in bioengineering is now receiving attention for
its potential use in medicine,191 from regulating gene expression 192, 193 to eliminating
antibiotic-resistance genes in bacteria.194, 195

Table 5: Applications for Metagenome Sequencing.

Applications

Reference

Discover rare microorganisms

196

Characterize unculturable microorganisms (“microbial dark matter”)

197

Study epidemiology of transmission events

198

Discover immune-escape variants and drug-resistance networks

199

Track evolutionary dynamics and reconstruct genome sequences

200

Discover gene products that are functional within microbial communities
but inactive or undetectable in monocultures

201

Characterize biological pathways to discover new chemical compounds

202

Discover compounds from bacterial communities in their natural habitats
with high-throughput in situ cultivation

203, 204

Identify CRISPR/Cas systems

205

189. Kitahara K., Yasutake Y. and Miyazaki K.
(2012) Mutational robustness of 16S ribosomal
RNA, shown by experimental horizontal genetransfer in Escherichia coli. Proc Natl Acad Sci
U S A 109: 19220-19225
190. Rajendhran J. and Gunasekaran P. (2011)
Microbial phylogeny and diversity: small
subunit ribosomal RNA sequence analysis andbeyond. Microbiol Res 166: 99-110
191. Polstein L. R., Perez-Pinera P., Kocak D.
D., Vockley C. M., Bledsoe P., et al. (2015)
Genome-wide specificity of DNA binding,gene regulation, and chromatin remodeling by
TALE- and CRISPR/Cas9-based transcriptional activators. Genome Res 25: 1158-1169
192. Polstein L. R. and Gersbach C. A. (2015) A
light-inducible CRISPR-Cas9 system for control of endogenous gene activation. Nat Chem
Biol 11: 198-200
193. Nihongaki Y., Kawano F., Nakajima T. and SatoM. (2015) Photoactivatable CRISPR-Cas9 for
optogenetic genome editing. Nat Biotechnol
33: 755-760
194. Yosef I., Manor M., Kiro R. and Qimron U.
(2015) Temperate and lytic bacteriophages
programmed to sensitize and kill antibiotic-resistant bacteria. Proc Natl Acad Sci U S A
112: 7267-7272
195. Skennerton C. T., Imelfort M. and Tyson G. W.
(2013) Crass: identification and reconstructionof CRISPR from unassembled metagenomic
data. Nucleic Acids Res 41: e105196. Lynch M. D. and Neufeld J. D. (2015) Ecology
and exploration of the rare biosphere. Nat Rev
Microbiol 13: 217-229
197. Panigrahi K., Applegate G. A., Malik G. andBerkowitz D. B. (2015) Combining a Clostridial
enzyme exhibiting unusual active site plasticity
with a remarkably facile sigmatropic rearrangement: rapid, stereocontrolled entry into densely
functionalized fluorinated phosphonates
for chemical biology. J Am Chem Soc 137:
3600-3609
198. Bryant J. M., Schurch A. C., van Deutekom
H., Harris S. R., de Beer J. L., et al. (2013)
Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome
sequencing data. BMC Infect Dis 13: 110
199. Li B., Yang Y., Ma L., Ju F., Guo F., et al. (2015)
Metagenomic and network analysis reveal
wide distribution and co-occurrence of environmental antibiotic resistance genes. ISME J
200. Spang A., Saw J. H., Jorgensen S. L.,
Zaremba-Niedzwiedzka K., Martijn J., et al.
(2015) Complex archaea that bridge the gap
between prokaryotes and eukaryotes. Nature
521: 173-179
201. Franzosa E. A., Hsu T., Sirota-Madi A.,
Shafquat A., Abu-Ali G., et al. (2015) Sequencing and beyond: integrating molecular ‘omics’
for microbial community profiling. Nat Rev
Microbiol 13: 360-372
202. Harvey A. L., Edrada-Ebel R. and Quinn R. J.
(2015) The re-emergence of natural products
for drug discovery in the genomics era. Nat
Rev Drug Discov 14: 111-129
203. Nichols D., Cahoon N., Trakhtenberg E.
M., Pham L., Mehta A., et al. (2010) Use ofichip for high-throughput in situ cultivation of“uncultivable” microbial species. Appl Environ
Microbiol 76: 2445-2450
204. Ling L. L., Schneider T., Peoples A. J.,
Spoering A. L., Engels I., et al. (2015) A new
antibiotic kills pathogens without detectable
resistance. Nature 517: 455-459
205. Skennerton C. T., Imelfort M. and Tyson G. W.
(2013) Crass: identification and reconstructionof CRISPR from unassembled metagenomic
data. Nucleic Acids Res 41: e105

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Reviews
Banerjee J., Mishra N. and Dhas Y. (2015) Metagenomics: A new horizon in cancer research. Meta Gene 5:
84-89
Doudna J. A. and Charpentier E. (2014) Genome editing. The new frontier of genome engineering withCRISPR-Cas9. Science 346: 1258096
van Dijk E. L., Auger H., Jaszczyszyn Y. and Thermes C. (2014) Ten years of next-generation sequencing
technology. Trends Genet 30: 418-426

Eukaryotes
Micro- and macro-eukaryotes are abundant, diverse, and ubiquitous in theenvironment.206 In the mammalian gut, these organisms impact the bacterial
environment, forming complex communities that impact health and disease.207
Culture-based methods, and even microscopy techniques, are usually unsuccessful
in identifying micro-eukaryotes due to their cryptic phenotypes.208 Fortunately, NGS is
not limited by phenotype, and it is improving our understanding about this enigmaticgroup, which still has many unknown sequences of uncharacterized genes. 209

Reviews
Richards S. (2015) It’s more than stamp collecting: how genome sequencing can unify biological research.
Trends Genet 31: 411-421
Grattepanche J. D., Santoferrara L. F., McManus G. B. and Katz L. A. (2014) Diversity of diversity: conceptualand methodological differences in biodiversity estimates of eukaryotic microbes as compared to bacteria.
Trends Microbiol 22: 432-437

References
Calderon-Norena D. M., Gonzalez-Novo A., Orellana-Munoz S., Gutierrez-Escribano P., Arnaiz-Pita Y., et al.
(2015) A single nucleotide polymorphism uncovers a novel function for the transcription factor Ace2 during
Candida albicans hyphal development. PLoS Genet 11: e1005152
Frykman P. K., Nordenskjold A., Kawaguchi A., Hui T. T., Granstrom A. L., et al. (2015) Characterizationof Bacterial and Fungal Microbiome in Children with Hirschsprung Disease with and without a History ofEnterocolitis: A Multicenter Study. PLoS One 10: e0124172
Heisel T., Podgorski H., Staley C. M., Knights D., Sadowsky M. J., et al. (2015) Complementary ampliconbased genomic approaches for the study of fungal communities in humans. PLoS One 10: e0116705
Hill J. A., O’Meara T. R. and Cowen L. E. (2015) Fitness Trade-Offs Associated with the Evolution of
Resistance to Antifungal Drug Combinations. Cell Rep
Linde J., Duggan S., Weber M., Horn F., Sieber P., et al. (2015) Defining the transcriptomic landscape of
Candida glabrata by RNA-Seq. Nucleic Acids Res 43: 1392-1406
Ma X., Baron J. L., Vikram A., Stout J. E. and Bibby K. (2015) Fungal diversity and presence of potentiallypathogenic fungi in a hospital hot water system treated with on-site monochloramine. Water Res 71: 197-206
O’Meara T. R., Veri A. O., Ketela T., Jiang B., Roemer T., et al. (2015) Global analysis of fungal morphology
exposes mechanisms of host cell escape. Nat Commun 6: 6741
Pi B., Yu D., Dai F., Song X., Zhu C., et al. (2015) A genomics based discovery of secondary metabolite
biosynthetic gene clusters in Aspergillus ustus. PLoS One 10: e0116089
Piotrowski J. S., Okada H., Lu F., Li S. C., Hinchman L., et al. (2015) Plant-derived antifungal agent poacic
acid targets beta-1,3-glucan. Proc Natl Acad Sci U S A 112: E1490-1497
Ramaprasad A., Mourier T., Naeem R., Malas T. B., Moussa E., et al. (2015) Comprehensive evaluationof Toxoplasma gondii VEG and Neospora caninum LIV genomes with tachyzoite stage transcriptome andproteome defines novel transcript features. PLoS One 10: e0124473

58

Microbes and metagenomics in human health206. Andersen L. O., Vedel Nielsen H. andStensvold C. R. (2013) Waiting for the humanintestinal Eukaryotome. ISME J 7: 1253-1255
207. Flegontov P., Votypka J., Skalicky T.,
Logacheva M. D., Penin A. A., et al. (2013)
Paratrypanosoma is a novel early-branchingtrypanosomatid. Curr Biol 23: 1787-1793
208. Scanlan P. D. and Marchesi J. R. (2008)
Micro-eukaryotic diversity of the human distal
gut microbiota: qualitative assessment using
culture-dependent and -independent analysis
of faeces. ISME J 2: 1183-1193
209. de Vargas C., Audic S., Henry N., Decelle J.,
Mahe F., et al. (2015) Ocean plankton. Eukaryotic plankton diversity in the sunlit ocean.
Science 348: 1261605

Hugerth L. W., Muller E. E., Hu Y. O., Lebrun L. A., Roume H., et al. (2014) Systematic design of 18SrRNA gene primers for determining eukaryotic diversity in microbial consortia. PLoS One 9: e95567
There is a need to generate adequate primer sets for broad taxonomy-range identification by 18S rRNA
gene high-throughput sequencing. The authors designed degenerate primers at every alignment position ofannotated eukaryotic 18S rRNA sequences. Primers were tested in silico, and the best-performing pairs were
further tested by amplicon sequencing of DNA from different sources. Regions V4 and V5 were the mostinformative and provided good agreement between tested samples and previous data from similar sources.
Primer combinations can generate highly discriminatory sequences by NGS. However, the best-performing
set reaches 79% accuracy at genus-level assignment, whereas 18S copy-number variation limits its use insemiquantitative approaches. Nevertheless, these primers can provide qualitative estimation of eukaryotic
communities in complex samples.
Illumina Technology: Nextera DNA Library Prep Kit, MiSeq

210. Franzosa E. A., Hsu T., Sirota-Madi A.,
Shafquat A., Abu-Ali G., et al. (2015) Sequencing and beyond: integrating molecular ‘omics’
for microbial community profiling. Nat Rev
Microbiol 13: 360-372
211. Wu A. R., Neff N. F., Kalisky T., Dalerba P.,
Treutlein B., et al. (2014) Quantitative assessment of single-cell RNA-sequencing methods.
Nat Methods 11: 41-46
212. Ning L., Liu G., Li G., Hou Y., Tong Y., et al.
(2014) Current challenges in the bioinformatics
of single cell genomics. Front Oncol 4: 7
213. Macaulay I. C. and Voet T. (2014) Single cell
genomics: advances and future perspectives.
PLoS Genet 10: e1004126

Calo S., Shertz-Wall C., Lee S. C., Bastidas R. J., Nicolas F. E., et al. (2014) Antifungal drug resistanceevoked via RNAi-dependent epimutations. Nature 513: 555-558

Single-Cell Sequencing (SCS)SCS can reveal functional differences that may be fundamental for pathogenicity,transmissibility, and antibiotic resistance.210 These differences may be hidden in
mixed-cell populations. In nonculturable microorganisms, where pure cultures areunavailable, SCS can produce complete genome, transcriptome, or plasmid datafrom a single cell. This technique can provide insights into the evolution, phylogeny,
and biological functions of “microbial dark matter” in human health.
In transcriptomics, single-cell RNA-Seq has the key advantage of resolving
biases introduced by a few cells with high gene expression that skew the average
expression level in the population.211 The main challenges of SCS include capturing
single cells, amplifying minimal amounts of nucleic acid, and analyzing data. There
has been rapid progress in the development of techniques and bioinformatic tools tomeet these challenges.212, 213
For comprehensive literature on single-cell DNA sequencing methods, refer to:• All Illumina publication reviews (http://www.illumina.com/science/publications/publications-review.html)• Illumina Methods Review (http://www.illumina.com/content/dam/illumina-marketing/documents/products/research_reviews/sequencing-methods-review.pdf)• Illumina Single-Cell Sequencing Review (http://www.illumina.com/content/dam/
illumina-marketing/documents/products/research_reviews/single-cell-sequencingresearch-review.pdf)

Reviews
Stegle O., Teichmann S. A. and Marioni J. C. (2015) Computational and analytical challenges in single-celltranscriptomics. Nat Rev Genet 16: 133-145
Wang Y. and Navin N. E. (2015) Advances and applications of single-cell sequencing technologies. Mol Cell
58: 598-609
Lasken R. S. and McLean J. S. (2014) Recent advances in genomic DNA sequencing of microbial species
from single cells. Nat Rev Genet 15: 577-584

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References
Wu A. R., Neff N. F., Kalisky T., Dalerba P., Treutlein B., et al. (2014) Quantitative assessment of singlecell RNA-sequencing methods. Nat Methods 11: 41-46
Accurate single-cell RNA-Seq is of interest to uncover expression patterns from precious samples and to
avoid biases introduced by nonrepresentative cells. The authors evaluated single-cell transcriptomes (fromhuman cultured cells) obtained with 2 different RNA amplification methods, followed by Nextera library prep
and sequencing with a HiSeq instrument. Single-cell transcriptomes were compared against qPCR and RNASeq of the bulk sample. Pooling single-cell data resembled the real bulk sample transcriptome. The authors
conclude that single-cell transcriptomes obtained by microfluidics yield accurate transcriptomes.
Illumina Technology: Nextera XT DNA Library Prep Kit, HiSeq 2000 150 bp PE reads

Plasmidome
Plasmids are self-replicating extrachromosomal genetic elements that may
horizontally transfer genes among microbes in a population.214, 215, 216 Metagenomes
contain DNA from the hosts but also the plasmids they carry. Due to the small sizeof plasmids, their DNA is overwhelmingly less abundant than host DNA. As a result,the DNA extraction methods and plasmid copy number are critical for obtaining an
accurate and complete plasmidome. Systems such as transposon-aided capture ofplasmids (TRACA)217 and multiple displacement amplification (MDA), coupled with
NGS, have improved the resolution of these elements.218

Reviews
Dib J. R., Wagenknecht M., Farias M. E. and Meinhardt F. (2015) Strategies and approaches in plasmidome
studies-uncovering plasmid diversity disregarding of linear elements? Front Microbiol 6: 463
Krupovic M. and Koonin E. V. (2015) Polintons: a hotbed of eukaryotic virus, transposon and plasmid
evolution. Nat Rev Microbiol 13: 105-115
Jorgensen T. S., Kiil A. S., Hansen M. A., Sorensen S. J. and Hansen L. H. (2014) Current strategies formobilome research. Front Microbiol 5: 750

References
Fu Y., Liu L., Li X., Chen Y., Jiang Y., et al. (2015) Spread of a common blaNDM-1-carrying plasmidamong diverse Acinetobacter species. Infect Genet Evol 32: 30-33
Carbapenem resistance is mediated by the gene New Delhi metallo-β-lactamase-1 (NDM-1). The geneis carried by plasmids that may have been transmitted by Acinetobacter to serious enteropathogens. The
authors characterized Acinetobacter isolates carrying the blaNDM-1 gene using PCR, 16S rRNA, andblaOXA-5 gene sequencing and biochemical methods. They identified a common novel plasmid widelydispersed in diverse Acinetobacter species. The common backbone structure indicates that this plasmid isvital in transfer of the blaNDM-1 gene among these different species.
Illumina Technology: HiSeq 2000
Nigro S. J., Holt K. E., Pickard D. and Hall R. M. (2015) Carbapenem and amikacin resistance on alarge conjugative Acinetobacter baumannii plasmid. J Antimicrob Chemother 70: 1259-1261
The authors used NGS to obtain the complete sequence of the extensively resistant A. baumannii isolate D46
and its plasmid pD46-3, which simultaneously transfers resistance to carbapenems and aminoglycosides.
The backbone of this plasmid resembles that of pD72-2 and pAb-G7-2 (repAci6 plasmids). It has acquired
the blaOXA-23 gene, indicating a role in dissemination of carbapenem resistance in A. baumannii.
Illumina Technology: HiSeq
Yamaichi Y., Chao M. C., Sasabe J., Clark L., Davis B. M., et al. (2015) High-resolution genetic analysisof the requirements for horizontal transmission of the ESBL plasmid from Escherichia coli O104:H4.
Nucleic Acids Res 43: 348-360
Relatively little is known about regulatory mechanisms for the dissemination of extended-spectrum betalactamases (ESBL). The authors used transposon insertion-site sequencing analysis (TnSeq)219 to identify

60

Microbes and metagenomics in human health214. Bleicher A., Schofl G., Rodicio Mdel R. andSaluz H. P. (2013) The plasmidome of a Salmonella enterica serovar Derby isolated frompork meat. Plasmid 69: 202-210
215. Brolund A., Franzen O., Melefors O.,
Tegmark-Wisell K. and Sandegren L. (2013)
Plasmidome-analysis of ESBL-producing
escherichia coli using conventional typing andhigh-throughput sequencing. PLoS One 8:
e65793216. Song X., Sun J., Mikalsen T., Roberts A. P.
and Sundsfjord A. (2013) Characterisation of
the plasmidome within Enterococcus faecalis
isolated from marginal periodontitis patients in
Norway. PLoS One 8: e62248
217. Jones B. V. and Marchesi J. R. (2007) Transposon-aided capture (TRACA) of plasmids resident in the human gut mobile metagenome.
Nat Methods 4: 55-61
218. Brown Kav A., Benhar I. and Mizrahi I. (2013)
A method for purifying high quality and highyield plasmid DNA for metagenomic and deep
sequencing approaches. J Microbiol Methods
95: 272-279
219. van Opijnen T. and Camilli A. (2013) Transposon insertion sequencing: a new tool forsystems-level analysis of microorganisms. Nat
Rev Microbiol 11: 435-442

genes that enable the maintenance and spread of pESBL, a plasmid isolated from E. coli O104:H4. Two
short regions constituted a novel regulatory system controlling the spread of this plasmid. Further, amethyltransferase encoded by pESBL protected pESBL from restriction in new hosts, suggesting it may playa role in expansion of host range.

220. Kopf M. and Hess W. R. (2015) Regulatory
RNAs in photosynthetic cyanobacteria. FEMS
Microbiol Rev 39: 301-315

Illumina Technology: MiSeq
Uchiya K., Takahashi H., Nakagawa T., Yagi T., Moriyama M., et al. (2015) Characterization of a novel
plasmid, pMAH135, from Mycobacterium avium subsp. hominissuis. PLoS One 10: e0117797
Mycobacterium avium subsp. hominissuis causes pulmonary disease in nonimmunosuppressed individuals andin pigs. The authors used NGS to obtain the complete genome sequence of plasmid pMAH135, derived from M.
avium isolated from an HIV-negative patient with pulmonary disease. pMAH135 contains sequence homology to
mycobactin biosynthesis proteins, type VII secretion system proteins, and putative domains for multidrug efflux
transporter. Genes carried by this plasmid were present more frequently in clinical pulmonary disease isolates
and absent in pigs. The authors conclude that this plasmid influences both pathology and host specificity.
Illumina Technology: HiSeq 2000 100 bp PE reads
Hamidian M., Kenyon J. J., Holt K. E., Pickard D. and Hall R. M. (2014) A conjugative plasmid carrying
the carbapenem resistance gene blaOXA-23 in AbaR4 in an extensively resistant GC1 Acinetobacterbaumannii isolate. J Antimicrob Chemother 69: 2625-2628
NGS sequencing was applied to the Australian A. baumannii A85 isolate (Global Clone 1), an extensively
resistant microbe harboring blaOXA-23 in Tn2006. Genes for resistance to older antibiotics are in the chromosome,in an AbaR3 resistance island. A second copy of the ampC gene in Tn6168 confers cephalosporin resistance,
and the gyrA and parC genes have mutations leading to fluoroquinolone resistance. An 86,335 bp repAci6
plasmid (pA85-3, carrying blaOXA-23 in Tn2006 in AbaR4) was shown to transfer resistance to imipenem,
meropenem, and ticarcillin/clavulanate into a susceptible recipient. A85 also contains 2 small cryptic plasmidsof 2.7 and 8.7 kb and carries a novel KL15 capsule locus and the OCL3 outer core locus.
Illumina Technology: HiSeq
Skarin H. and Segerman B. (2014) Plasmidome interchange between Clostridium botulinum,
Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into
distinctly different pathogens. PLoS One 9: e107777
Clostridium novyi sensu lato contains the closely related species C. botulinum, C. novyi, and C. haemolyticum
with different pathogenic phenotypes associated with extrachromosomal mobile elements. WGS was
performed in 24 representative strains of Clostridium novyi sensu lato and their respective plasmidomes. In
addition, 4 Clostridium novyi sensu lato lineages and a large variable plasmidome composed of 13 plasmidgroups (PG1 to PG13) were obtained. PG1 are unstable prophages encoding the BoNT gene cluster and theC3 toxin. The plasmids PG3, PG4, and PG6 can carry genes for C2 toxin, phospholipase C, and epsilon toxin
B, while PG8-PG11 contains α-toxin-encoding prophages. There was modest correlation between species
and lineages, perhaps due to the limited mobility of toxin-encoding plasmids and phages between lineages.
This observation implies complex genetic relationships between these species: plasmids and phages, which
may have been modularly exchanged, can create the pathogenic characteristics of these lineages.
Illumina Technology: TruSeq DNA Library Prep Kit, HiSeq 2000 100 bp PE reads

Transcriptome Sequencing
Transcriptome sequencing provides information on the activation of genes andbiological pathways. This information provides insight into what activities the
microbes are engaged in, to answer the question: “What are they doing?” Whentranscriptome sequences are compared to their genome sequences, it is possible
to map and characterize genes, regulatory elements, and operon structures. For
example, metatranscriptomic analysis revealed that small RNA (sRNA) encoded inintergenic regions can have multiple regulatory functions in archaea and bacteria.220
Metatranscriptomic analysis can reveal complex interactions between community
members to make efficient use of shared resources.

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Combining transcriptomics with additional experimental techniques can provide
detailed insight into pathogenic processes and the specific pathways involvedin transmission and disease.221 This approach is of great utility in geneticallyhomogeneous microbes, such as Salmonella enterica serovar Enteritidis, wheretranscriptomic profiles can discern pathways associated with different pathogenic
phenotypes.222 There is a growing interest in the role of pathogens in cancer research,
sparked by the discovery of pathogen transcriptomic signatures in tumors. The
discovery highlights the role of the immune response and inflammation in hostpathogen interactions.223

Reviews
Creecy J. P. and Conway T. (2015) Quantitative bacterial transcriptomics with RNA-seq. Curr Opin Microbiol
23: 133-140
Miyakoshi M., Chao Y. and Vogel J. (2015) Regulatory small RNAs from the 3’ regions of bacterial mRNAs.
Curr Opin Microbiol 24: 132-139

References
Yost S., Duran-Pinedo A. E., Teles R., Krishnan K. and Frias-Lopez J. (2015) Functional signaturesof oral dysbiosis during periodontitis progression revealed by microbial metatranscriptome analysis.
Genome Med 7: 27
Periodontal disease is common worldwide, and current models hold oral microbiome dysbiosis accountablefor periodontitis progression. The authors used metatranscriptomic and metagenomic analysis of microbialcommunities in subgingival biofilms to identify changes in community composition and expression networks.
Global metabolic signatures were consistent with disease progression by upregulation of virulence factors
from organisms not associated with disease and from recognized periodontal pathogens. Diseaseprogression cannot be solely attributed to recognized pathogen virulence factors but to changes incommunity metabolic signatures that favor the expression of disease-causing genes. This study highlights theimportance of transcriptomic studies in understanding periodontal disease.
Illumina Technology: Nextera XT DNA Library Prep Kit, MiSeq v2 150 bp PE reads
Shah D. H. (2014) RNA sequencing reveals differences between the global transcriptomes ofSalmonella enterica serovar enteritidis strains with high and low pathogenicities. Appl Environ
Microbiol 80: 896-906
S. enterica serovar Enteritidis (SE) causes bacterial food-borne gastroenteritis mainly transmitted by chicken
eggs and poultry products. Strains are phenotypically diverse and vary in their virulence potential, despite
their genetic homogeneity. RNA-Seq was used to characterize in vitro differences in global transcriptomes of
SE strains with high and low pathogenicity at avian body temperature. The study identified 252 differentiallyexpressed genes, mostly downregulated and related to transcriptional regulators, virulence, and motility in
low-pathogenicity strains. This study provides a concise view of transcriptional signatures associated withpathogenicity levels in SE. These signatures may be targets of functional characterization studies, due to theirpotential role in virulence.
Illumina Technology: TruSeq RNA Library Prep Kit, HiSeq 2000 v3
Edlund A., Yang Y., Yooseph S., Hall A. P., Nguyen D. D., et al. (2015) Meta-omics uncover temporal
regulation of pathways across oral microbiome genera during in vitro sugar metabolism. ISME J
Kernell Burke A., Guthrie L. T., Modise T., Cormier G., Jensen R. V., et al. (2015) OpaR controls a network of
downstream transcription factors in Vibrio parahaemolyticus BB22OP. PLoS One 10: e0121863
Kopf M., Klahn S., Scholz I., Hess W. R. and Voss B. (2015) Variations in the non-coding transcriptome as adriver of inter-strain divergence and physiological adaptation in bacteria. Sci Rep 5: 9560
Papenfort K., Forstner K. U., Cong J. P., Sharma C. M. and Bassler B. L. (2015) Differential RNA-seq of Vibrio
cholerae identifies the VqmR small RNA as a regulator of biofilm formation. Proc Natl Acad Sci U S A 112:
E766-775
Franzosa E. A., Morgan X. C., Segata N., Waldron L., Reyes J., et al. (2014) Relating the metatranscriptome
and metagenome of the human gut. Proc Natl Acad Sci U S A 111: E2329-2338

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Microbes and metagenomics in human health221. Rosinski-Chupin I., Sauvage E., Sismeiro O.,
Villain A., Da Cunha V., et al. (2015) Single
nucleotide resolution RNA-seq uncovers new
regulatory mechanisms in the opportunistic
pathogen Streptococcus agalactiae. BMC
Genomics 16: 419
222. Shah D. H. (2014) RNA sequencing reveals
differences between the global transcriptomesof Salmonella enterica serovar enteritidis
strains with high and low pathogenicities. Appl
Environ Microbiol 80: 896-906
223. Chen Y. and Wei J. (2015) Identification of
Pathogen Signatures in Prostate Cancer Using
RNA-seq. PLoS One 10: e0128955

GLOSSARY OF TERMS
AL

anthropogenic litter

antagomir

A small synthetic RNA that is perfectly complementary to
the specific miRNAAs

arsenic

Cryosols

Frozen soil within 1 meter of the surface and characterized
by waterlogging during periods of thaw

DDT

dichlorodiphenyltrichloroethane.

Endosymbionts

Smaller symbiotic partners living inside a host organism

HMP

Human Microbiome Project

HPV

Human papillomavirus

iHMP

Integrative Human Microbiome Project

MERS-CoV

Middle East respiratory syndrome coronavirus

MMETSP

Marine Microbial Eukaryote Transcriptome
Sequencing Project

NGS

Next-generation sequencing, also called high-			
throughput sequencing

PIT

particle interceptor traps

reassortment

Recombination of genomic material, especially as it 		
occurs naturally in related viruses

SARS

Severe acute respiratory syndrome

SARS-CoVSARS-associated coronavirusSbantimony

tropism

Viral tropism is the specificity of a virus for a particular 		
host tissue

WBD

White Band disease

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Scientific Publication Reviews can be accessed atwww.illumina.com/pubreviews

Immunology Research Review
Repertoire sequencing has enabled researchers to identify
unique receptor variants found in individuals with susceptibility
to hematological malignancies, autoimmune diseases, andallergen response.
Illumina next-generation sequencing provides the quality,
throughput, and read lengths required by the researchcommunity to map the human immune response at highresolution. The emergence of new approaches such asphase-defined sequencing and single-cell sequencing canbe expected to accelerate this knowledge base.

Single Cell Research Review
Most of the impetus for single-cell tissue sequencing has comefrom cancer research, where cell lineage and the detectionof residual disease are of paramount concern. The same
approaches are being used to improve our understandingof massively complex biological systems, such as neuraldevelopment and immunology.
This document highlights recent publications that demonstrate
the use of Illumina technology for single-cell sequencing andvery low input applications and techniques.

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Pub. No. 1270-2015-004 Current as of 16 November 2015