An Introduction to Illumina Next-Generation
Sequencing Technology for Agriculture
Deciphering DNA sequences is essential for virtually all branches of biological research. Capillaryelectrophoresis (CE)-based sequencing has enabled scientists to elucidate genetic informationfrom almost any organism or biological system. Although this technology has become widelyadopted, inherent limitations in throughput, scalability, cost, speed, and resolution can hinder
scientists from obtaining essential genomic information. To overcome these barriers, an entirely
new technology was developed—next-generation sequencing (NGS), a fundamentally different
approach to sequencing that has triggered numerous ground-breaking discoveries. The years since
the introduction of NGS have seen a major transformation in the way scientists extract genetic
information from biological systems, revealing insight about the genome, transcriptome, andepigenome. This introduction will highlight the benefits of using NGS for agricultural research.

www.illumina.com/agricultureWelcome to Next-Generation Sequencing
Advances in NGS have been instrumental in advancing scientific fields from human disease research
to environmental and evolutionary science. NGS lends itself particularly well to the agriculturallaboratory, where genomes can be complex and a priori knowledge scarce. NGS allows identificationof novel SNPs accounting for valuable phenotypic traits or the sequencing of unculturable organisms.
For veterinary diagnostic applications, single-base resolution allows tracking of microbial or viral
adaptation over short periods of time, both in the laboratory and in the environment.
As evidence of the uptake of this technology, NGS data output has increased at a phenomenal rate,
more than doubling each year since it was introduced. In 2007, a single sequencing run could produce
about one gigabase (Gb) of data. By 2012, that rate had jumped 1000× to one terabase (Tb) of data
in a single sequencing run. With this tremendous increase in output has come a 105-fold decrease in
the cost of bacterial whole-genome sequencing. In 1995, sequencing the 1.8 megabase (Mb) genomeof Haemophilus influenzae with CE technology cost ~1 million US dollars, taking over one year. Today,
sequencing the 5 Mb genome of Escherichia coli with Illumina NGS can be done in one day at afraction of the cost.

Basic Concepts of NGS
In principle, NGS is similar to Sanger-based, or CE sequencing. The bases of a small fragment of
DNA are sequentially identified from signals emitted as each fragment is re-synthesized from a DNA
template strand. NGS extends this process across millions of reactions in a massively parallel fashion.
This advance enables rapid sequencing of many large stretches of DNA, with the latest instruments
capable of producing 1 Tb of data in a single sequencing run.
To illustrate how this process works, consider a single genomic DNA (gDNA) sample. The gDNA isfirst fragmented into a library of small segments and sequenced. The newly identified strings of bases,
called reads, are then reassembled bioinformatically using a known reference genome (resequencing),
or assembled together using advanced computational techniques if no reference genome is available(de novo sequencing). The full set of aligned reads reveals the entire genomic sequence of the sample(Figure 1). Once the sample library is prepared, all of the sequencing steps through data analysis canbe performed on a single instrument, facilitating rapid turnaround with minimal hands-on time.
Figure 1: Next-Generation Sequencing Steps

A

gDNA

B

Library Prep

C

D

E

G
G
G

A
A
A

C T
C
C T
C T

G

A

C T

Parallel Sequencing

Alignment

G...G A C T G...

G
G
Sequence Data

A. Extracted gDNA.
B. Library preparation fragments genomic DNA and adds adapters to generate a library.
C. DNA fragments within the library are each sequenced in parallel.
D. Individual sequence reads are reassembled by aligning to a reference genome.
E. The whole genome sequence is derived from the consensus of aligned reads.

Library Preparation
How NGS is used experimentally is largely dictated by the way sequencing libraries are prepared andthe way the data are analyzed, with the actual sequencing steps remaining fundamentally unchanged.
A growing number of library preparation kits are currently available to provide complete reagents andprotocols for sequencing whole genomes, small genomes, mRNA, targeted regions such as wholeexomes, targeted or enriched regions, protein-binding regions, and more. To address specific research
objectives, researchers have developed many novel protocols to isolate specific regions of the genome
associated with a given biological function.
Library preparation protocols for NGS are generally more rapid and straightforward than those forCE-based Sanger sequencing. With NGS, researchers can start directly from a gDNA or cDNA library.
The DNA fragments are then ligated to specific oligonucleotide adapters needed to perform thesequencing biochemistry, requiring as little as 90 minutes with Illumina’s Nextera® technology (Figure 2).
In contrast, CE-based Sanger whole-genome sequencing requires genomic DNA to be fragmented firstand cloned into either bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs).
Then, each BAC/YAC must be further subcloned into a sequencing vector and transformed into theappropriate microbial host. Template DNA is then purified from individual colonies or plaques prior tosequencing. This process can take days or even weeks to complete.
Figure 2: NGS Library Preparation Overview

Adapters

DNA

Sequencing-Ready Library

NGS library preparation starts directly from fragmented genomic DNA. Platform-specific oligonucleotide adapters, needed to performthe sequencing biochemistry and index samples, are ligated to each end of the fragments to yield the sequencing-ready library.

Scalable Studies Enabled by Multiplexing
For veterinary diagnostic applications such as sequencing small bacterial or viral genomes,
researchers can choose to use a lower output instrument and process a smaller number of samples
per run, or opt for a higher output instrument to process a large number of samples. Multiplexing
enables large numbers of samples to be simultaneously sequenced during a single experiment(Figure 3). To accomplish this, individual “barcode” sequences are added to each sample so they canbe differentiated during the data analysis.
With multiplexing, NGS dramatically reduces the time to data for large numbers of samples.
Processing hundreds of amplicons on hundreds of samples using CE technology generally requires
several weeks or months. The same number of samples can now be sequenced in a matter ofhours and fully analyzed within two days using NGS. With highly automated, easy-to-use protocols,
researchers can go from experiment to data to publication faster, more easily, and more costeffectively than ever before.

Figure 3: Sample Multiplexing Overview

A

B

C

O

D

Sample 1

Sample 2

A. Two representative DNA fragments from two unique samples, each attached to a specific barcode sequence that identifies the
sample from which it originated.
B. L
 ibraries for each sample are pooled and sequenced in parallel. Each new read contains both the fragment sequence andits sample-identifying barcode.
C. Barcode data are used to de-multiplex, or differentiate reads from each sample.
D. Each set of reads is aligned to the reference sequence.

Paired-End Sequencing
Paired-end (PE) and Mate Pair (MP or long-insert, paired-end) sequencing, where both ends of a DNA
fragment are sequenced (Figure 4), allow long range positioning of the DNA fragment. Because thedistance between each paired read is known, alignment algorithms can use this information to precisely
map the reads, resulting in superior alignment across difficult-to-sequence regions or junctions near
repetitive genome regions, such as those found in many plant (wheat and maize) genomes. Illumina
NGS offers the flexibility of variable insert sizes (typically 400 bp to 5 kb) and read lengths (35–300 bp),
allowing high resolution characterization of any genome.
Figure 4: Paired-End Sequencing and Alignment

Paired-End Reads

Alignment to the Reference Sequence

Read 1
Reference
Repeats

Read 2Paired-end sequencing enables both ends of the DNA fragment to be sequenced. Because the distance between each paired readis known, alignment algorithms can use this information to map the reads over repetitive regions more precisely. This results in muchbetter alignment of the reads, especially across difficult-to-sequence or repetitive regions of the genome.

Analyze, Store, and Share in the Cloud
Data analysis is an important factor to consider for sequencing applications. One of the biggest
challenges with NGS systems has been the requirement for a high-performance computing
infrastructure, enterprise-level storage, and highly skilled bioinformatics and IT staff. Dependingon the application, most subsequent analyses can be run directly on optimized software installedon the sequencer’s internal computer, or in BaseSpace®, Illumina’s unique cloud computing
environment. By storing and analyzing data in the cloud, BaseSpace users can instantly sharedata with collaborators across the hallway or across the globe. The BaseSpace Application Store
will provide seamless access to a wide variety of commercial software tools, as well as collectionof well-known and open-source algorithms from academic institutions.

End-to-End Solution
Only Illumina NGS provides a fully supported solution from DNA to results, with specialized library prep
choices for the applications you are working on, robust and proven sequencing reagents, and a wide
range of simple data analysis tools (Figures 5 and 6).
Figure 5: Illumina’s End-to-End NGS Workflow on the MiSeq System

Prep

Sequence

AnalyzeShare

15 minutes hands-on

20 minutes hands-on

fully automated, on
instrument or in cloud

Secure,unlimited storage

1.5
HOURS

4

HOURS

3

HOURS

INSTANTLYNGS workflow on the MiSeq system includes library preparation, massively parallel sequencing, automated data analysis,
and cloud-enabled data analysis, storage, and sharing.

Figure 6: Illumina’s End-to-End NGS Workflow on the NextSeq 500 System

Prep

Sequence

AnalyzeShare

5 hours hands-on

20 minutes hands-on

5 minutes hands-on,
on-site or in cloud

Secure,unlimited storage

1.5

29

DAYS

HOURS

5

HOURS PER SAMPLEINSTANTLYThe NextSeq 500 system workflow delivers highly accurate sequencing data. Data analysis includes alignment and variant calling.

Agricultural Applications
Until recently, sequencing an entire genome was a major endeavor. The ability of NGS to produce alarge volume of data in a short period of time makes it a powerful tool for whole-genome sequencing.
Its speed and cost-effectiveness also make it an ideal method for targeted sequencing, which focuses
on defined regions of the genome.
To accelerate the return on investment in breeding practices, there’s an increasing drive towards lower
cost methods that will enable the implementation of SNP genotyping tools for routine screening in
more agriculture crops. The recent evolution of sequence-based genotyping methods is a direct resultof the pressing need for more cost-effective solutions.

Whole-Genome Sequencing
Whether a research project is focused on a novel species or just one that has never been investigatedbefore using genetic tools, de novo sequencing is a first step toward understanding the genetic
underpinnings of a plant or animal’s functions and its interaction with the environment. NGS is fully
scalable, capable of sequencing large (wheat and oats), medium (bovine, porcine, and ovine), small(rice), and tiny genomes (viral and bacterial). By contrast, whole-genome sequencing using CE-based
Sanger technology requires significant time and resources, even for small genomes. The power andspeed of NGS was demonstrated during the 2011 enteroaggregative E. coli outbreak in Europe, which
prompted a rapid scientific response. Using NGS data, researchers were able to quickly generate ahigh-quality, whole-genome sequence of the bacterial strain, enabling them to better understand thegenetic mutations conferring the increased virulence.
NGS has also transformed metagenomics, enabling the study of large microbial communities directlyin their natural environment without prior culturing. These studies can yield important information about
the complex and diverse populations of microbes associated with animal and plant development, fromrumen flora that enhance animal digestion to root-associated bacteria involved in nitrogen fixation.

Example: De novo Sequencing
One challenge associated with sequencing several plant and animal genomes is the lack of reference
genomes available for many species. This means that whole-genome sequencing must often bedone de novo, where the reads are assembled without aligning to a reference sequence. Paired-end
reads and increasing reads lengths up to 300 bp help produce longer contigs for de novo sequencing
by filling gaps in the consensus sequence, resulting in a more complete assembly. The resulting
assembled genome can be used to assign map positions and stack diverse breed information,
enabling subsequent resequencing to discover SNPs and other genetic variations.

Targeted Sequencing
With targeted sequencing, only a subset of genes or defined regions in a genome are sequenced,
allowing researchers to focus time, expenses, and data storage resources on the most impactful regionsof the genome for their research. Amplicon sequencing refers to sequencing selected regions of the
genome spanning hundreds of base pairs up to several kilobases. For bovine studies, the Illumina
TruSeq® Custom Amplicon Library Preparation kit allows researchers to perform rapid in-solutionamplification of custom-targeted regions from genomic DNA. Using this approach, thousands ofamplicons spanning multiple samples can be simultaneously prepared and indexed in a matter of hours.
With the ability to process numerous amplicons and samples on a single run, NGS enables researchers
to simultaneously analyze all genomic content of interest in one experiment, at fraction of the time andcost of conventional CE sequencing.

Example: 16S Metagenomic Sequencing
A common use of amplicon sequencing involves comparing the bacterial 16S rRNA gene, a widelyused method for studying phylogeny and taxonomy in prokaryotes. This method has been used toevaluate bacterial diversity in many environments, allowing researchers to characterize microbiomes
from samples that are otherwise difficult or impossible to study. NGS, with its ability to sequence
thousands of organisms in parallel, is uniquely suited to this application. The ability to pool samples
and obtain high sequence coverage during a single run allows NGS to identify rarer variants that are
missed, or too expensive to identify, using CE-based sequencing approaches. Table 1 illustrates thecomparison of 96 samples done on the MiSeq versus CE sequencing for the purpose of comparison.
Table 1: Illumina NGS and CE-Based Sanger Sequencing for Targeted Applications
Parameter

MiSeq System

Sanger Sequencing

Samples in project

96

96

Number of amplicons

1212Target panel size

~5 kb

~5 kb

Time for library prep

< 3 hours< 3 hours

Sequencing time

1 day

6 daysPrice per amplicon*$1 USD$4 USD

Project price*< $2000 USD> $4500 USD

Coverage depth per amplicon> 13,000 x

2 x**

On instrument data analysis?

Yes

No

* Excluding PCR amplification.
** Including bidirectional sequencing.

True metagenome sequencing studies, comprising hundreds or thousands of possible genomes, arecost- and labor-prohibitive with CE sequencing, and only possible with high-throughput NGS systems
such as the Illumina HiSeq®, NextSeq™, and MiSeq® systems.

Next-Generation Genotyping (NGG)
Implementing SNP genotyping tools for routine screening in many agriculture crops relies upon
low cost genetic screening methods. The lower the cost, the more researchers can accelerate thereturn on investment in breeding practice for genetic mapping, screening backcross lines, purity
testing, constructing haplotype maps, and performing association and genomic evaluation for plantagrigenomics studies. We use NGG to refer to genotyping using NGS sequencing methods. NGG
encompasses methods to discover novel SNPs and genotype them, often simultaneously in many
individuals or specimens. For small genomes such as Drosophila or high profile research species
like Arabidopsis, most NGG methods can be completed with standard library preparations andmanufacturer multiplexing protocols. Target enrichment or reduction of genome complexity can be
employed for larger genomes, requiring homebrew library preparation and optimized multiplexing toensure low cost, while offering sufficient overlap in sequencing coverage to call SNPs reliably. NGG
benefits for agricultural research vary slightly by method and offer lower cost, reduced ascertainment
bias, and in some cases, an ability to perform comparative analysis across samples in the absenceof a reference genome. Proven NGG methods include PCR-based approaches (amplicon or longrange PCR), restriction enzyme methods (RAD-Seq or published GBS methods by Cornell), andtargeted methods of enrichment to capture desired regions of the genome, such as microsatellites,
exome, and methylation sensitive sequencing.

Example: RAD-Seq
Restriction site-associated DNA sequencing (RAD-Seq) is a genome complexity reduction method,
designed to reliably interrogate a fraction of a target genome instead of the entire genome sequence.
This method involves use of a tailored restriction enzyme protocol optimized to characterize SNPs
and sequencing depth to meet a given project goal. The short fragments (or RAD tags) of genomic
DNA that flank the enzyme recognition site are screened for the presence of genetic variation. NGS
compatible RAD fragment libraries enable massively parallel and multiplexed sample sequencing of
RAD-tag libraries, facilitating the rapid discovery and genotyping of tens to hundreds of thousands ofidentical SNPs across large populations.

Discover. Develop. Deploy.
The advent of NGS has enabled agricultural researchers to study livestock, crops, and biologicalsystems at a level never before possible. With clear benefits over Sanger-based CE sequencing,next-generation sequencing can accelerate and enhance agricultural research, advance thedevelopment of high-value trait screening methods, and enable the swift deployment of theseapplications in the real word. To identify the sequencing platform that is optimal for your researchneeds, visit www.illumina.com.

From Innovation to Publication
As NGS technology continues to evolve, researchers are making fascinating discoveries in a number ofbiological fields, unlocking answers never before possible in all fields of research. As a result, there has
been an explosion in the number of peer-reviewed scientific publications, including over 4,500 featuring
Illumina sequencing technology. Selected recent examples relevant to agriculture are listed below.
Whole-Genome Sequencing
1. Larkina DM, Daetwylerb HD, Hernandezc AG, Wrightc CL, Hetrickc LA, et al. (2012) Whole-genome resequencing of two elite sires for thedetection of haplotypes under selection in dairy cattle. Proc Natl Acad Sci U S A. 109:7693–7698.
2. The International Barley Genome Sequencing Consortium (2012) A physical, genetic and functional sequence assembly of the barley genome.
Nature: 10.1038/nature11543. [Epub ahead of print].
3. Bannantine JP, Wu C, Hsu C, Zhou S, Schwartz DC, et al. (2012) Genome sequencing of ovine isolates of Mycobacterium avium
subspecies paratuberculosis offers insights into host association. BMC Genomics 13:89.
4. Huang X, Feng Q, Qian Q, Zhao Q, Wang L, et al. (2009) High-throughput genotyping by whole-genome resequencing.
Genome Res 19: 1068–1076.
5. Metzger ML (2010) Sequencing technologies—the next generation. Nat Rev Genet. 11: 31–46.

De novo Sequencing
6. Liu M, Qiao G, Jiang J, Yang H, Xie L, et al. (2012) Transcriptome sequencing and de novo analysis for ma bamboo(Dendrocalamus latiflorus Munro) using the Illumina platform. PLoS ONE 7: e46766.
7. Lulin H, Xiao Y, Pei S, Wen T, Shangqin H (2012) The first Illumina-based de novo transcriptome sequencing and snalysis of safflower flowers.
PLoS ONE 7: e38653.
8. Dejun Li, Zhi Deng, Bi Qin, Xianghong Liu. and Zhonghua Men (2012) De novo assembly and characterization of bark transcriptome using
Illumina sequencing and development of EST-SSR markers in rubber tree (Hevea brasiliensis Muell. Arg.).BMC Genomics 13:192.

Metagenomics
9. Kava AB, Sassona G, Jamia E, Doron-Faigenboima A, Benharb I, et al. (2012) Insights into the bovine rumen plasmidome.
Proc Natl Acad Sci U S A.109:5452–5457.
10. Avrani S, Wurtzel O, Sharon I, Sorek R and Lindell D (2011) Genomic island variability facilitates Prochlorococcus-virus coexistence.
Nature 474: 604–608.

Targeted Sequencing
11. Cronn R, Knaus BJ, Liston A, Maughan PJ, Parks M, et al. (2012) Targeted enrichment strategies for next-generation plant biology.
Am J Bot. 99: 291–311.

Next-Generation Genotyping
12. Baird N, Etter P, Atwood T, Currey M, Shiver A, et al. (2008) Rapid SNP discovery and genetic mapping using sequenced RAD markers.
PloS One 3:e3376.
13. Poland JA, Brown PJ, Sorrells ME, Jannink J-L (2012) Development of high-density genetic maps for barley and wheat using a novel
two-enzyme genotyping-by-sequencing approach. PLoS ONE 7: e32253
14. Elshire RJ, Glaubitz JC, Sun Q, Poland JA, Kawamoto K, et al. (2011) A robust, simple genotyping-by-sequencing (GBS) approach forhigh diversity species.PloS One 6: e19379.
15. Kirst M, Resende M, Munoz P, and Neves L. (2011) Capturing and genotyping the genome-wide genetic diversity of trees for association
mapping and genomic selection. BMC Proceedings 5: I7.
16. Peterson BK, Weber JN, Kay EH, Fisher HS, and Hoekstra HE (2012) Double digest RADseq: an inexpensive method for de novo SNP
discovery and genotyping in model and non-model species. PLoS One 7: e37135.

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