Application Note: Sequencing

De Novo Assembly and Resequencingof the Saccharomyces cerevisiae Genome
An end-to-end workflow for sequencing small genomes, from library prep to data analysis in theBaseSpace® environment.
Figure 1: Illumina’s End-to-End Solution for Sequencing Small Genomes

DNA Extraction

Library Preparation

Sequencing

De Novo
AssemblyDNASTAR
SeqMan NGenAlignment
45 minutes

120 minutes

39 hours

The Broad’s IGV

Introduction

Library Construction

Saccharomyces cerevisiae is one of the most studied eukaryotic
model organisms, representing all the basic gene functions foreukaryotes. In collaboration with White Labs, Inc. (San Diego, CA),
Illumina sequenced eight closely related S. cerevisiae strains on theMiSeq® system to capture their biological diversity and gain insight
into the differences among the strains. These differences include
copy number and ploidy variations, genome rearrangements, andpolymorphism changes. The data generated in this study represents
a significant contribution to ongoing yeast research and is available
for public access on the BaseSpace cloud computing platform. Thisapplication note describes a complete workflow (Figure 1), fromDNA extraction to simple de novo genome assembly using SeqMan
NGen software from DNASTAR1 (Madison, WI) within the BaseSpace
environment.

Genomic DNA libraries were prepared using the Nextera® XT Library
Preparation Kit, which provides a rapid, easy, and efficient workflow
optimized for small genomes (Figure 2). By simultaneously fragmenting
and tagging the DNA with adapters (a process called “tagmentation”),
this protocol enables the preparation of sequencing-ready libraries inless than 90 minutes.

Methods and Results
Genomic DNA Isolation
Yeast cultures were obtained through collaboration with White Labs.
Genomic DNA from the eight yeast cultures was isolated using theMasterPure™ Yeast DNA Purification Kit from Epicentre, an Illumina
company2 (Madison, WI). This kit employs a non-enzymatic approachto cell lysis, followed by protein precipitation and subsequent nucleic
acid isolation. This process results in high yields of purified, highmolecular-weight DNA. After isolation, the purified DNA was quantified
using fluorimetric methods and diluted to the optimal concentration forlibrary construction.

The Nextera transposome tagmented 1 ng of yeast DNA, resulting
in adapter-ligated DNA fragments. The tagmented DNA was purified
and then amplified by PCR before cluster generation and sequencing.
Because the Nextera XT Library Preparation Kit uses bead-based
sample normalization, library quantification was not necessary before
pooling. Thus, the kit provides a streamlined workflow from library
preparation to sequencing.

Sequencing
Sequencing took advantage of the flexibility and throughput affordedby the MiSeq system’s on-board cluster generation. For rapid genome
sequencing, 5 µl aliquots from each of the eight strains were pooled
together, loaded onto a MiSeq reagent cartridge, and clustered on theMiSeq instrument. Samples were sequenced using 2 × 250 bp pairedend reads and the FASTQ MiSeq Reporter workflow. Sequencing
generated a total of 18 million reads, resulting in an average coverage
depth of 39× per sample. Base call (*.bcl) files generated by the MiSeq
system were converted to FASTQ files and uploaded automatically tothe BaseSpace environment for secondary analysis.

Application Note: Sequencing
Figure 2: Nextera XT Library Preparation

Alignment Using Resequencing Pipeline andIntegrative Genomics Viewer

Transposomes
Genomic DNA~ 300 bp

Tagmentation

The kit uses a “tagmentation” reaction—simultaneous fragmentation and adapter-tagging of DNA—to enable fast and efficient library
preparation. Indices and motifs ~requiredfor clustering and sequencing
300 bpare added during subsequent low-cycle amplification.

P5
Index 1

Data Analysis

Read 1 Sequencing Primer

Read 2 Sequencing Primer

Index 2

Data analysis poses a challenge for any microbiologist analyzing
Amplification
next-generation sequencing data. ThePCR
BaseSpaceplatform provides asolution for storage and streamlined analysis, with ready-to-use
software applications.
P7

p5

Index1 Rd1 SPRd2 SP Index2

p7

Sequencing-Ready Fragment

Read Assembly Using De Novo Pipeline andSeqMan NGen Software
For this application, Illumina used SeqMan NGen de novo
assembly software, which allows quick and accurate assembly ofeither single-read or paired-end data. Sequencing data from theyeast samples were imported into SeqMan NGen and reads wereassembled defining the parameters as 350 bp for the pair distancesand 12 Mb for the estimated genome size. Across the eight samples,an average N50 length of 63,246 bp was obtained. These draft
genome assemblies are ready to use in a range of analyses, including
gene correlations, genome rearrangements, or copy numbervariations. The assembly metrics for each sample are shown in Table
1. All strains were compared with the same de novo parameters,with no changes in function of size or coverage. For furtherinvestigation, the estimated genome size should be considered forbetter assemblies, because some strains have variable ploidy andgenome size.

The data were also analyzed using the resequencing pipeline of theMiSeq system, which uses the Burrows-Wheeler Algorithm (BWA) tocreate alignment (*.bam) files. These files can then be opened andvisualized using the Integrative Genomics Viewer3 (IGV), a secondary
analysis application developed by the Broad Institute and available
on the BaseSpace platform. IGV allows researchers to align BAM
files to a reference genome. Three of the S. cerevisiae samples wereselected for analysis of variations in general, with an emphasis onchromosome 9 (Table 2). In the samples labeled 2591 and 2542,
phenotypically, one flocculates normally while the other does not.
Flocculation refers to the ability of yeast to aggregate and form fibrousinterconnections, or clumps. Comparison between the flocculation
genes FLO1 and FLO8 did not reveal significant genetic changes,
but the FLO11 gene was shown to have an insertion of 1,000 bp
in sample 2542 (Figure 3). This gene encodes a glycolipid (GPIanchor) that can be attached to the C-terminus of a protein during
post-translational modification. To understand whether the observed
insertion can affect the strain’s ability to flocculate, further analysis
is required.

Conclusions
The workflow outlined in this application note—including DNA
extraction from culture samples, sequencing, and simple data
analysis—is broadly applicable to the analysis of any small genome.
The data obtained from S. cerevisiae strains is available for public
use on the BaseSpace platform4. As new applications are developed
within the BaseSpace environment, the analysis can be extended
to other areas of microbiology research. The high accuracy andresolution of the sequencing workflow allows researchers to sequence
the genomes of bacteria, archaea, fungi, or lower eukaryotes with aconvenient and easy-to-use assembly pipeline.

Table 1: Assembly Output for S. cerevisiae Strains

Sample ID

Base Count

N50

Number ofContigs

Clusters Passing
FilterCoverage Basedon 12 Mb

Location2521

10,146,523

27K

1571

1,018,471

21

England2535

22,353,560

35K

1035

7,395,956

154

San Diego, USA

2542

21,913,088

30K

739

1,189,456

25

Belgium

2543

30,685,603

27K

1551

1,258,847

26

Belgium

2545

39,965,87034K

1493

1,286,525

27

Belgium

2590

21,367,407

62K

523

1,602,957

33

—*

2591

21,870,148

55K

615

1,803,178

38

California, USA

2592

22,222,588—*—*

2,957,468

62

England*Information not available.

Application Note: Sequencing
Figure 3: Alignment to Reference Genome

Genome assemblies from two samples were aligned to the S. cerevisiae reference genome. A region of chromosome 9 is shown here.

Learn More
Go to www.illumina.com/miseq to learn more about the next revolutionin personal sequencing. For more information about the use of Illuminasequencing technology in microbial genomics, visit

Table 2: BWA Whole-Genome
Sequencing Output

Sample ID

Number ofReads% Coverage2542

2.378,912

93%

98%

References

2590

1,602,957

54%

88%

1.

www.dnastar.com

2591

3,606,356

94%

98%

2.

www.epibio.com

3.

www.broadinstitute.org/igv

4.

www.basespace.illumina.com/projects/White_Labs

www.illumina.com/microbiology.

Illumina • 1.800.809.4566 toll-free (U.S.) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com
FOR RESEARCH USE ONLY
© 2013, 2014 Illumina, Inc. All rights reserved.
Illumina, IlluminaDx, BaseSpace, BeadArray, BeadXpress, cBot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy,
Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iSelect, MiSeq, Nextera, NuPCR, SeqMonitor, Solexa,TruSeq, TruSight, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registeredtrademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners.
Pub. No. 1270-2013-005 Current as of 11 November 2014

% Aligned