Design Content | Prepare Library | Sequence | Analyze Data

Microbial whole-genome sequencing withIllumina DNA PCR-Free Prep, Tagmentation
Fast, flexible library preparation for uniform genomic coverage for microbial species.

Introduction

Methods

Next-generation sequencing (NGS) has been established as animportant tool for analyzing small genomes (≤ 5 Mb), including
bacteria, viruses, and other microbes. Microbial NGS, including wholegenome sequencing (WGS) and targeted resequencing, enables
mapping and de novo assembly of novel organisms, completing
genomes of known organisms, and comparing genomes acrosssamples. However, PCR-dependent library preparation for microbial
WGS, particularly when combined with in-solution adapter tagging andfragmentation, can introduce bias, leading to uneven coverage acrossregions of the genome, especially regions with extreme or unevenbase composition.
To address this challenge, Illumina DNA PCR-Free Prep, Tagmentation(Illumina DNA PCR-Free) offers a unique combination of On-Bead
Tagmentation with a PCR-free workflow (Figure 1). Tagmentation
is a transposome-mediated reaction that combines tagging andDNA fragmentation into a single, rapid reaction. By removing PCRamplification steps and using bead-based chemistry, Illumina DNA
PCR-Free chemistry provides highly accurate sequence information for
sensitive applications, such as microbial WGS.
This application note demonstrates the exceptional performanceof Illumina DNA PCR-Free as part of a comprehensive workflow for
genome assembly of microbial organisms and presents sequencing
results for three different bacterial species with varying GC content.

Samples
DNA from three bacterial species with differing GC content (Table 1)
were obtained from the American Type Culture Collection (ATCC):
B. cereus strain 971 (ATCC, Catalog no. 14579D-5), E. coli strain
MG1655 (ATCC, Catalog no. 700926D-5), and R. sphaeroides strain
ATH 2.4.1 (ATCC, Catalog no. 17023D-5).
Table 1: GC content of sequenced microbial genomes
B. cereus

E. coliR. sphaeroides

Genome size

~5.4 Mb~4.6 Mb~4.1 Mb% GC content

~35%

~51%

~69%

Library preparation
Libraries were prepared from 200 ng of input bacterial genomic DNA(gDNA) using Illumina DNA PCR-Free reagents and the standardworkflow. For comparison, 600 ng of bacterial gDNA was shearedby Covaris ultrasonication and input into the TruSeq™ DNA PCR-Free
workflow. Sonication and the TruSeq DNA PCR-Free workflow weremodified to select for an average insert size of 450 bp.

Sequencing
Pooled libraries were sequenced on a MiSeq™ or NextSeq™ 550
Sequencing System with a run configuration of 2 × 150 bp.

Prepare Library

Sequence

Analyze Data

Illumina DNA PCR-Free

MiSeq, NextSeq 550 Systems

BaseSpace Sequence HubDRAGEN Germline App﻿Figure 1: Illumina DNA PCR-Free workflow—Illumina DNA PCR-Free is part of a comprehensive, streamlined workflow for microbial WGS that includes library
preparation, sequencing, and data analysis.

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Data analysis

Uniform coverage across genomes with varying
GC content

After sequencing was complete, data was streamed directly from theinstrument into the cloud ecosystem for push-button analysis using
DRAGEN™ apps available through BaseSpace™ Sequence Hub. TheDRAGEN Germline App v3.4.5 was used for reference alignment.

ResultsTo evaluate the performance of Illumina DNA PCR-Free for microbialWGS, results were compared to those obtained using TruSeq DNA
PCR-Free.

Robust library yield with lower input requirements

4000001.43500001.23000001.02500000.8

2000000.61500000.4

100000

0.2

50000
00
10

2030

4050

60

7080

90

100% GC content

3E. coli

1.8
1.6

3500003000001.42500001.2
1.02000000.81500000.6

100000

0.4

50000

0.2

Relative no. of bins
at %GC

4000102030

40

50

60

7080

90

100% GC content

R. sphaeroides

Normalized coverage

1.821350000

1.63000001.42500001.2
1.02000000.81500000.6

100000

0.4

50000

0.2

Relative no. of bins
at %GC

Library yield (nM)

B. cereus

1.60Normalized coverage

5Illumina DNA PCR-Free
TruSeq DNA PCR-Free

TruSeq DNA PCR-Free
Illumina DNA PCR-Free
Illumina DNA Prep (PCR+)
No. of bins

Relative no. of bins
at %GC

Illumina DNA PCR-Free results in equivalent or better total library yieldcompared to TruSeq DNA PCR-Free for the microbial species tested(Figure 2). Of particular note, library preparation with B. cereus gDNA
(an AT-rich genome) resulted in significantly higher yield. Importantly,Illumina DNA PCR-Free achieves similar library yields with a lower DNA
input requirement, as compared to TruSeq DNA PCR-Free (Table 2).

Normalized coverage

To assess coverage performance across bacterial species with a rangeof low, medium, and high GC content, normalized coverage datafrom Illumina DNA PCR-Free and TruSeq DNA PCR-Free were plottedagainst reference genome content by GC percentage. Both kits showeven coverage levels across all microbial species tested, regardlessof GC content (Figure 3, Table 2). For comparison, genome coverage
was also evaluated with Illumina DNA Prep (includes PCR). Results
show comparable performance with some biases at extremes of GCcontent (Figure 3). However, Illumina DNA Prep performs better thanlibrary prep kits with in-solution tagmentation.

000102030405060708090

100% GC content

0B. cereus

E. coliR. sphaeroides

Figure 2: Comparison of library yields across microbial genomes of varying
GC content—Illumina DNA PCR-Free generates sequencing libraries withequivalent or better yields, as compared to TruSeq DNA PCR-Free.

Figure 3: Comparison of read coverage across microbial genomes ofvarying GC content—Illumina DNA PCR-Free (blue line) provides consistent andcomparable read coverage across microbial genomes of varying GC content,
as compared to TruSeq PCR-Free (gray line) and Illumina DNA Prep (orangeline). The bell curve trace (black line) shows the actual GC composition of eachmicrobial species.

Table 2: Genome build metrics
B. cereus

E. coliR. sphaeroides

Illumina DNAPCR-FreeTruSeq DNA
PCR-FreeIllumina DNAPCR-Free

TruSeq DNA
PCR-FreeIllumina DNA
PCR-Free

TruSeq DNA
PCR-Free

200 ng

600 ng200 ng

600 ng200 ng

600 ng

1M

1M

1M

1M

1M

1M

Yield

4.5 nM

0.35 nM

3.9 nM

3.1 nM

2.7 nM

2.4 nM

Average alignment coverage over genome

52.2

51.0

61.1

62.5

58.7

59.9

Uniformity of coverage (% > 0.2 mean) over genome

98.9%

99.0%

99.3%

99.4%

96.9%

96.9%

Percent mapped

96.3%

93.1%

97.2%

97.8%

92.4%

93.0%

Library preparation
Input amountPaired-end (PE) reads subsampled

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TruSeq DNA PCR-Free
Library prep with adapter ligation and index tagging

Manual library quant andnormalization

Manualpooling

5 hr

2 hr

0.5 hrIllumina DNA PCR-Free, gDNA
Library prep with PCR-free
BLT tagmentation

Pool byvolume1.5 hr

0.5 hrFigure 4: Comparison of PCR-Free workflows—Illumina DNA PCR-Free delivers a rapid total assay time of 90 minutes from tagmentation through library clean-up,
compared to > 7 hours total assay time for TruSeq DNA PCR-Free.

On-Bead Tagmentation and PCR-free protocol

Learn more

Illumina DNA PCR-Free provides a unique and powerful combinationof benefits from On-Bead Tagmentation and PCR-free chemistry.
On-Bead Tagmentation uses bead-linked transposomes to perform
a more uniform tagmentation reaction compared to in-solution
tagmentation. After the bead-linked transposomes are saturated
with DNA, no additional tagmentation can occur, delivering robust
insert size control and normalized yields from DNA input amounts
above 150 ng.1,2 This minimizes quantification steps both before
and after library prep. Normalized libraries can be pooled by volume,thus avoiding time-consuming quantification of individual libraries.
By eliminating quantification and PCR steps, Illumina DNA PCR-Freeoffers significant time savings, as compared to TruSeq DNA PCR-Free(Figure 4).

To learn more about Illumina DNA PCR-Free, visit www.illumina.com/
products/by-type/sequencing-kits/library-prep-kits/dna-pcr-free-prep.
html

References
1.
2.

Illumina (2018). Nextera DNA Flex Library Preparation Kit. Accessed April
10, 2020.
Bruinsma S, Burgess J, Schlingman D, Czyz A, Morrell N, et al. Beadlinked transposomes enable a normalization-free workflow for NGS library
preparation. BMC Genomics. 2018;19(1):722.

SummaryIllumina DNA PCR-Free offers a unique combination of benefits from
On-Bead Tagmentation and PCR-Free chemistry steps. Illumina DNA
PCR-Free delivers exceptional ease of use, uniform coverage, andhigh-accuracy data for microbial WGS, even for challenging genomic
regions with extreme GC content.

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