Technical Note
ResolveDNA MicrobiomeAuthors
Jon Zawistowski, PhD
Senior Director
Research & Development
BioSkryb Genomics

Jeff Blackinton, PhD
Director
New Products & Services
BioSkryb Genomics

Durga Arvapalli, PhD
Scientist
Research & Development
BioSkryb Genomics

Isai Salas-Gonzalez, PhD
Scientist
Computational Biology
BioSkryb Genomics

Genomic Analysis of Single Bacteria using ResolveDNA
Microbiome
Bacteria make up the bulk of life on earth, and it is estimated that humans are aware of only asmall percentage of microbes. Even with our limited knowledge of bacterial diversity aroundus, ground breaking scientific discoveries have been made possible because of the bacteria
around us, including Taq polymerase, restriction endonucleases, and CRISPR gene editing
technology. Additionally, the more we learn about the symbiotic relationship between humans
and bacteria, the more we appreciate the profound impact bacteria have on human health.
Major advances have been made studying bacterial populations and selected strains. However,for technical reasons, it previously has been difficult to investigate bacterial heterogeneity
on a whole genome scale. BioSkryb Genomics has developed a novel product, ResolveDNA
Microbiome, which utilizes Primary Template-directed Amplification (PTA) technology toamplify DNA from a single bacterium with quality rivaling bulk DNA. This technology unlocks
the near-complete genome of a single bacterium, enabling myriad new applications, including,but not limited to, understanding diversity within bacterial strains, within environments, andnovel discovery of difficult-to-culture specimens (Figure 1).

Jay A.A. West, PhD

President, CEO, Co-founder
BioSkryb Genomics

Novel GeneDiscovery

Bacteriophage
Detection

Novel DrugResistance
Discovery

Bacterial
Population
Heterogeneity

ResolveDNA™
Microbiome

Novel
EnzymeDiscovery

Novel
Bacterial
Species
Discovery

New
Antibiotic
Moieties

Horizontal
Gene
Transfer

Figure 1. ResolveDNA Microbiome: Bacteria populate virtually every ecosystem known. Only a small fraction of bacterial species are characterized. The diversity of bacteria influence and shape their environments, and have signficant
human health impacts. ResolveDNA Microbiome can address a diverse set of biological questions that can lead tosignificant insights and impact.
For Research Use Only. Not for use in diagnostic procedures.

ResolveDNA Microbiome

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SingleBacteria
Isolation

Bacteria
Cell lysis

WGA

BeadPurification

Library
Preparation

Next
Generation
Sequencing

BaseJumper
Analysis

Figure 2. ResolveDNA Microbiome workflow. Single cells are
first isolated by FACS, followed
by efficient lysis of either gram
positive or gram negative bacteria. Lysed cells, containing ~5-10
fg of genomic DNA then undergo Primary Template-directed
Amplification. Amplified DNA
from the reaction is then purified and quantified, followed byligation or tagmentation-based
library preparation, sequencing,and analysis with BaseJumper.

Introduction

Materials and Methods

Microorganisms, the most diverse group of life on Earth1 play
crucial roles in the biochemical cycling on earth. They were thefirst life forms to appear on Earth, and are present in virtually
all habitats on the planet. Bacteria grow in soil, acidic hot
springs, radioactive waste, water, and deep in the Earth’s crust.
In addition, they grow in organic matter and the live bodies ofplants and animals, and scientists are increasingly appreciating
both the symbiosis and pathogenesis between bacteria andhuman health.

Single Bacteria Isolation. Three sample types were isolated
1) gram negative bacteria (E.coli), 2) gram positive bacteria(B. subtilis), and 3) a mixture of these species, to evaluate theperformance of the ResolveDNA Microbiome WGA Kit.

Beyond understanding our environment and the microbesthat inhabit it, microbiology has a long and storied historyof driving our understanding of fundamental biology, andof developing biological tools. Virtually all of molecularbiology owes the development of its manipulatory methods
to microbiology research, from restriction endonucleases
to PCR to CRISPR. Taken together, it is clear that delineating
microbial biodiversity2 is key to not only predicting ecosystem
responses to environmental changes3, but also improving
bioprocesses, discovering novel biological mechanisms andtools, understanding human health, and the elucidation of
new therapeutic biomolecules.
In the eukaryotic realm, the development of single-cell analysis
methodology over the past decade has revolutionized ourunderstanding of the heterogeneity and interconnectednessof the cellular microenvironment4-6. For largely technicalreasons, this revolution has not extended in the bacterial realm
to the same degree. Since the crux of each cell is its genome,
the capability to further resolve the bacterial diversity relies on
the ability to decipher the individual genomes of these cells.
Up until now, a bottleneck was the keystone technology tofaithfully prepare the single bacterial cell for next generation
sequencing analysis. To enable this capability we havedeveloped the ResolveDNA microbiome system to allow
amplification, library creation and sequencing analysis ofindividual bacterial cells (Figure 2). The ability to interrogate
the genomes of individual bacteria allows a wide expansionof application development in the discovery of new functional
elements of the microbiome7-8.

Bacterial stocks of each species were grown in LB broth for
24 hours at 37oC. Afterwards, cultures were filtered through a
20 µm filter to remove large cell clusters and counted by OD
measurement, then an aliquot of each sample was mixed tocreate a 1:1 cell ratio. Samples were centrifuged, resuspended
in 1X dPBS, and live/dead stained using Syto9/PI. Singlecell bacteria were sorted into 96-well plates containing 1 µL
ResolveDNA Cell Buffer using a Sony SH800 sorter equippedwith a 130um sorting chip. Sorted plates were briefly vortexed
and flash frozen and stored at -80C until ready to perform PTAWGA.
DNA Amplification with the ResolveDNA Microbiome WGAKit. The ResolveDNA Microbiome protocol was followed,
resulting in amplified bacterial DNA. This DNA was thenpurified with the ResolveDNA bead purification kit. We foundindividual bacteria typically yielded ~100-200 ng of amplified
DNA (Figure 3A) and had an average size range of 900 bases(Figure 3B). Purified amplified DNA was then transformed intosequencing libraries using the ResolveDNA Library Preparation
kit. Sequencing libraries were again purified and analyzed by
TapeStation 4200 (Agilent) which demonstrated optimal sizeof PTA amplified single bacterial genome libraries (~500bp)(Figure 3C). These libraries were then sequenced using theIllumina MiniSeq Platform. Raw sequencing data were analyzed
for quality, contig assembly, and alignment.

ResultsAnalysis of single bacteria demonstrated the ability to detect
nearly the entire bacterial genome from each individualcell. We found both bacteria species tested approached thetheoretical maximum genome size (Figure 4 Column A&B). For
this analysis approximately 20 of each bacteria were sequenced(5 representative shown - figure 4). We noted minor differencesin coverage, single cell genome of E. coli typically covered ~90-95% of the genome, where single B. subtilis detected up toFor Research Use Only. Not for use in diagnostic procedures.

ResolveDNA Microbiome3A)

B)

C)

Figure 3. ResolveDNA Microbiome single bacterial cell amplification and library preparation: isolated single bacteria were amplified, followed by bead based amplified DNA purification using the ResolveDNA bead purification system. We found the majority of single bacterial cells generated approximately 150 ng of amplified genomic DNA(A). To determine the size range of the amplification products were subjected to D5000 tapestation analysis (B). We found the range of amplification products
centered ~900 bp, while the overall range appeared to extend from 100 to ~1500 BP, which is slightly smaller than observed for eukaryotic cells. Amplified DNA (~ 100ng)
were then converted to sequencing libraries using th ResolveDNA library preparation kit. Insert size was then verified by TapeStation using the D1000 tape cassette.
Libraries were then subjected to NGS analysis using the Illumina MiniSeq using a high output 300 cycle kit, generating 150 Bp, paired end reads.

(A)

(B)

(C)

100% of the theoretical genome. The data further detected minor
components of other (unidentified) contigs, however these areconsidered to be minor contaminants, which based on the data do
not affect the ability to identify the species/genus of origin.
Having sequenced these individual bacteria, a third set of samples
was prepared where a combination of both the E. coli and B. subtilis
were sorted into wells (Figure 4C). Again, greater than 20 samples
were processed (4 representative samples shown). We found bothbacterial families were detectable, with cumulative contigs thelength of the respective genomes. In many cases it appears wedispensed more than one cell into each well, as both E. coli and B.
subtilis genomes were detected, however, in the last panel only B.
subtilis was detected, suggesting assay specificity. Trace amountsof human DNA and unidentified species DNA were detected inthese samples, however the short contigs make these reads easy toidentify and remove.
Collectively, the data provide evidence that minute numbers ofbacteria can be separated, amplified and prepared for NGS analysis.
Moreover, we demonstrate in a single well, multiple bacteria and theirrespective genomes can be differentiated using the ResolveDNA
next generation sequencing system.

Conclusions
BioSkryb has developed a powerful approach to investigating
diversity in the bacterial kingdom. Using the ResolveDNA Bacterial
WGA kits, microbial diversity can be routinely assessed with thehighest degree of resolution, the single bacterial cell. This enables
the capability to delineate mixed populations and recover the entire
genomes of these cells, to generate complete genomic profilesof unculturable bacteria, and to reveal the unique biology of thediversity of bacterial life.

Figure 4. ResolveDNA Microbiome performance. Individual bacterial cells were isolated by FACS, and amplified using the ResloveDNA Microbiome system. Amplified products were converted to sequencing libraries, and sequenced on an Illumina MiniSeq @ ~1 million reads/cell. The data demonstrate that > 90% of the single
cell genomes of (A) E. coli and (B) B. subtilis cells are able to be reproducibly detected. In addition, mixed species experiments (C) further demonstrate the ability todiscriminate multiple bacterial species in the same well.
For Research Use Only. Not for use in diagnostic procedures.

ResolveDNA Microbiome

4References:
1.
2.
3.
4.
5.
6.
7.
8.

Turnbaugh, P.J., et al., The human microbiome project. Nature, 2007. 449(7164): p.
804-10.
Chitsaz, H., et al., Efficient de novo assembly of single-cell bacterial genomes fromshort-read data sets. Nat Biotechnol, 2011. 29(10): p. 915-21.
Torsvik, V., L. Øvreås, and T.F. Thingstad, Prokaryotic diversity--magnitude, dynamics,and controlling factors. Science, 2002. 296(5570): p. 1064-6.
Ortega, M.A., et al., Using single-cell multiple omics approaches to resolve tumor
heterogeneity. Clin Transl Med, 2017. 6(1): p. 46.
de Bourcy, C.F., et al., A quantitative comparison of single-cell whole genome
amplification methods. PLoS One, 2014. 9(8): p. e105585.
Paolillo, C., E. Londin, and P. Fortina, Single-Cell Genomics. Clin Chem, 2019. 65(8): p.
972-985.
Chase, J.M. and J.A. Myers, Disentangling the importance of ecological niches fromstochastic processes across scales. Philos Trans R Soc Lond B Biol Sci, 2011. 366(1576):p. 2351-63.
Ofiteru, I.D., et al., Combined niche and neutral effects in a microbial wastewater
treatment community. Proc Natl Acad Sci U S A, 2010. 107(35): p. 15345-50.

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All data on file.
For Research Use Only. Not for use in diagnostic procedures.
BIOSKRYB and RESOLVEDNA are trademarks of BioSkryb, Inc.
All other product names and trademarks are the property of
their respective owners.
TAS.006, 101/2022