Prep are Lib rary | Seq uence | Analyze Data

Direct Bacterial Colony Sequencing with the
Nextera™ DNA Flex Library Preparation Kit
Eliminate the need for separate DNA isolation and high-accuracy quantitation steps with thestreamlined, cost-effective Flex Direct Colony Method.
Introduction
Traditional methods used in microbiology and public healthlaboratories to characterize bacterial pathogens can be timeconsuming and complex. After a bacterial pathogen is isolated on a
primary culture plate, it must be identified to provide an early
prediction of the potential disease progression and to determine theantibiotic resistance profile of the pathogen. These procedures arefollowed by antibiotic susceptibility testing to identify significant
resistance mechanisms and acquired drug resistances. Fortunately,
next-generation sequencing (NGS) technologies offer considerable
benefits to researchers and clinicians by reducing the time and effort
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needed to identify and characterize microbial samples. , With NGS
in the laboratory, bacterial whole-genome data can be obtained andbacterial characterization can be performed with a single sequencing
run.
Though whole-genome sequencing (WGS) has provided significant
advantages in the speed, accuracy, and depth of information
12
available to microbiology labs, , the preparation of bacterial samples
for sequencing has remained labor-intensive. NGS libraries for WGS
are typically prepared from bacterial samples that are subcultured
followed by separate genomic DNA extraction protocols. In somecircumstances, however, it is advantageous to sequence as soon as
possible; for example, after colonies appear on primary isolation
plates. Sequencing directly from bacterial colonies eliminates theextra time and cost resulting from subculture and DNA extraction
steps. Sequencing directly from colonies also increases the overall
speed and efficiency of the workflow and could be vital to early
infection control interventions. To address these challenges in
microbiology and public health laboratories, Illumina offers the
Nextera DNA Flex Library Preparation Kit along with the Flex Direct
Colony Method—an innovative library prep method that supports
quick and easy library preparation directly from bacterial colonies.
This application note demonstrates the Flex Direct Colony Method
using eight microbial pathogens.

Nextera DNA Flex library prep advantages
Featuring unique chemistry that integrates multiple pre- and postlibrary preparation steps, the Nextera DNA Flex Library Preparation Kit
delivers the fastest workflow with the fewest number of steps in the
Illumina portfolio. In addition to speed and efficiency, it offers
exceptional flexibility for sample input type, input amount, and a wide
range of supported applications. The Nextera

For Research Use Only. Not for use in diagnostic procedures.

Figure 1: Nextera on-bead transposome chemistry—(A) Bead-linked
transposomes mediate the simultaneous fragmentation of gDNA and the additionof Illumina sequencing primers. (B) Reduced-cycle PCR amplifies sequencing
ready DNA fragments and adds indexes and adapters. (C) Sequencing-readyfragments are washed and pooled.

DNA Flex Library Prep Kit enables DNA extraction directly frombacterial colonies, resulting in additional time and cost savings while
improving data consistency.35
A major advance in Illumina library prep chemistry and key feature of
the Nextera DNA Flex Library Preparation Kit is On-Bead
Tagmentation, which uses bead-linked transposomes to mediate
simultaneous DNA fragmentation and tagging of Illumina sequencing
primers (Figure 1).

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Table 1: Eight bacterial species used to test the Flex Direct Colony Method
Gram type

Genome size

GC content

DNA in 5 µl sample

Pseudomonas aeruginosa

Negative

6.3 Mb

66.20%

13.7 µg

Klebsiella pneumoniae

Negative

5.7 Mb

57.10%

18.4 µgEnterobacter cloacae

Negative

5.5 Mb55.10%

8.9 µgEscherichia coli

Negative

5.2 Mb50.80%

11.3 µg

Acinetobacter baumannii

Negative

4.0 Mb

39.00%

8.8 µgEnterococcus faecalis

Positive

3.3 Mb37.30%

1.9 µgStreptococcus agalactiae

Positive

2.1 Mb

35.00%

2.2 µgStaphylococcus aureus

Positive

2.8 Mb32.90%

8.1 µgOn-Bead Tagmentation provides several significant advantages:lEliminates the need for highly accurate quantitation of the initial
DNA sample, across a wide DNA input range (100–500 ng), saving
time and costs associated with DNA quantitation kits andequipment.

lEliminates the need for a separate DNA fragmentation step, saving
time and costs associated with separate shearing instruments or
enzymatic kits.

lEliminates the need for highly accurate quantitation of individuallibraries before normalization, across a wide DNA input range(100–500 ng), before pooling and sequencing.

Table 2: Library preparation kits and methods
Library preparation kit

ProtocolDNA input

Nextera DNA Flex Library Prep Kit

Flex Standard Method~200 ng

Nextera DNA Flex Library Prep Kit

Flex Low-Input Method

1 ng

Nextera DNA Flex Library Prep Kit

Flex Direct Colony Method

10 µl lysate

Nextera XT DNA Library Prep Kit

Nextera XT Method

1 ng

On-Bead Tagmentation produces libraries with consistent insert sizes(~350 bp) over a wide DNA input range.6 This wide range of DNA input(100–500 ng) offers increased flexibility for varying sample types,
including precious samples. This new chemistry delivers robust
performance with DNA input amounts down to 1 ng. With ≥ 100 ng
DNA input, the On-Bead Tagmentation reaction becomes saturated,leading to consistent, normalized yields.6 This normalized input range
offers significant flexibility in the amount of input DNA used for
Nextera DNA Flex library prep.

Methods
Flex Direct Colony Method
In brief, the Flex Direct Colony Method begins with the collection of 5 µlof bacterial sample (half a 10 µl loopful) from blood agar plates. The
bacterial sample is then processed through a short protocol including
bead beating and solid-phase reversible-immobilization (SPRI)–based
cleanup steps (Figure 2). For an in-depth description of the Flex Direct
Colony Method, see the Nextera DNA Flex Microbial Colony Extraction
Guide.

Quantification of DNA from colony loop samples
To show that 5 µl of colonies contains more than sufficient DNA tosaturate the beads (> 100 ng), a retrospective DNA quantification of
the lysates was performed using material that had been processed
through bead beating, spinning, SPRI-based cleanup steps. DNA
content was quantified by Qubit 3.0 Fluorometer (Thermo Fisher
Scientific).

For Research Use Only. Not for use in diagnostic procedures.

Figure 2: Nextera DNA Flex Direct Colony Method—The Flex Direct Colony
Method is a rapid, 5-step protocol where crude lysates are prepared from bacterialcolonies for direct input into the Nextera DNA Flex Library Preparation Kit. Solidphase reversible immobilization (SPRI) beads are used in many molecular biology
kits for DNA clean-up steps.

Sequencing library preparation
Sequencing libraries were prepared from eight bacterial species
chosen based on their pathogenic profile, mixture of Gram positive
and Gram negative type, and their diverse range of GC content(Table 1). To compare library prep performance, four library
preparation workflows were performed (Table 2).

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Figure 3: DNA content in Flex Direct Colony Method lysates—Left Y-axis shows DNA concentrations of lysates prepared using the Flex Direct Colony Method. All 24 lysate
samples were measured (8 bacterial species × 3 replicates). DNA concentrations range from 18.5–185 ng/µl. Right Y-axis illustrates index representation of the sequenced
libraries.

Libraries were prepared as described in the Nextera DNA Flex Library
Prep Reference Guide or the Nextera XT DNA Library Prep Reference
Guide. The Nextera XT Method, the Flex Low-Input Method, and theFlex Standard Method, used DNA purified from colonies by the
DNeasy UltraClean Microbial Kit (QIAGEN, Cat. No. 12224-50). The
Nextera XT and Flex Low-Input Methods used 1 ng of purified DNA,accurately quantified by Qubit 3.0 Fluorometer. For the Flex Standard
Method, the purified DNA samples were quantified by Qubit 3.0 andimprecisely diluted to approximately 200 ng each—which is well
above the 100 ng bead saturation threshold. The Flex Direct Colony
Method used lysates generated as described in the Microbial Colony
Extraction protocol. All libraries were prepared in triplicate.

Sequencing
All libraries were sequenced on a NextSeq™ 550 System using
NextSeq 500/500 v2 High Output Kits (Illumina, Cat. No. FC-4042004) with a run configuration of 2 × 150 bp to generate sufficientgenomic coverage for de novo assembly. Base calling and quality
scoring were performed with onboard NextSeq Control Software 7 andReal-Time Analysis v27 software.

Data analysis
Index representation plots were generated in BaseSpace™ Sequence
Hub, the Illumina genomics computing platform. Genome coverageplots were calculated with Artemis8 across the reference genomes forthree of the eight microorganisms tested. High, medium, and low GC
content pathogens were selected: Pseudomonas aeruginosa (66.2%),
Escherichia coli (50.8%), and Staphylococcus aureus (32.9%). All
sequencing data sets were down-sampled to 4 million reads perorganism.

For Research Use Only. Not for use in diagnostic procedures.

To compare de novo genome assembly quality, the metrics N50,
number of contigs, and largest contig were calculated using SPAdes
v3.9.09 and QUAST10 for all eight microorganisms, using the 4 million
read, down-sampled data sets.

Results
Quantification of DNA content in colonies
All eight microorganisms and their replicates yielded DNA ranging
from 18.5–185 ng/µl post-SPRI—based clean-up (Figure 3). This
represents a coefficient of variance (CV) of 49% for the DNA input into
the Flex Direct Colony Method, and a final CV of 19.6% for index
representation postsequencing. This lower CV postsequencing
indicates that highly consistent and accurate results were obtained,even with libraries that were generated from an initially wide DNA
input range.

Index representation and library insert size comparisons
Good index representation indicates that each library, when
multiplexed together with other libraries, receives roughly equal
representation of coverage on the flow cell. This translates into more
uniform coverage and accuracy of results for a given experiment. To
assess index representation of all four library prep methods, thepercent index representation was measured and plotted for all 24
libraries (8 bacterial samples × 3 replicates) for each library prep
method (Figure 4). With the Flex Direct Colony Method, the index
representation was highly uniform with a CV of 19.6% across all 24
libraries. Furthermore, the data reveal a dramatic improvementcompared to the Nextera XT DNA workflow, which showed a CV of
36.7% across the 24 libraries for index representation (Figure 4D).

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Figure 4: Comparison of index representation—The bar graphs illustrate the index representation of the 24 libraries prepared with the (A) Flex Standard, (B) Flex Direct
Colony, (C) Flex Low-Input, and (D) Nextera XT Methods. CV = the coefficient of variance across all 24 libraries pooled and sequenced together on the same flow cell.

Figure 5: Comparison of insert sizes—The bar graphs illustrate the insert sizes of the 24 libraries prepared with the (A) Flex Standard, (B) Flex Direct Colony, (C) Flex LowInput, and (D) Nextera XT Methods. CV = the coefficient of variance across the insert sizes of all 24 libraries.

For Research Use Only. Not for use in diagnostic procedures.

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Figure 6: Comparison of whole-genome coverage —The plots show a 4 Mb genome view of coverage across the full genome for three bacterial libraries prepared with four
different library preparation methods: (A) Pseudomonas aeruginosa, (B) Escherichia coli, and (C) Staphylococcus aureus. Data sets for each library were down-sampled to
4 million reads.

Comparison of library insert sizes across the library replicates andDNA sample types provides a measure of the consistency andreliability of the library prep methods. To assess the library prep
consistency of all four library prep methods, insert sizes were plotted
for all 24 libraries (Figure 5). All Nextera DNA Flex methods
demonstrated highly consistent and uniform insert sizes. The Flex
Direct Colony Method produced a 3% CV on the median insert size
across all 24 samples. These results show how the bead-saturating
feature of the Nextera DNA Flex Kit successfully eliminates the needto quantify DNA with high accuracy before library preparation.

For Research Use Only. Not for use in diagnostic procedures.

The data also reveal how On-Bead Tagmentation provides amechanism for the preparation of exceptionally uniform insert sizes(Figure 5A-C). Compared to the Nextera XT Method, which requires
accurate DNA quantitation steps prelibrary preparation and additional
quantitation steps for library normalization postlibrary preparation, allthree Nextera DNA Flex Methods delivered significantly better insert
size uniformity with lower CV values (Figure 5A-C). Even the Flex LowInput Method, where the DNA does not saturate the beads and thus atightly normalized yield is not expected, outperforms the Nextera XT
Method. The Low-Input method delivered a CV of 2% for medianinsert size with post library prep, equivolume pooling only (Figure 5).

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Figure 7: Comparison of genome assembly metrics—Bar graphs illustrate de novo genome assembly metrics for all 24 libraries prepared with four different library
preparation methods. Metrics include (A) Total number of contigs, (B) N50 values, and (C) Largest contig.

These data illustrate that high-accuracy DNA quantification steps
before and after library preparation are no longer needed with the
Nextera DNA Flex Library Prep Kit. The best results were obtained (CVof 12.3% for sample representation and 1% for median insert size)with the Nextera DNA Flex Standard protocol, where a saturating
amount of purified DNA was used (Figure 4A and Figure 5).

Comparison of whole-genome coverage
Beyond assessments of library prep performance, the sequencing
data sets generated from the four Nextera methods were analyzed tocompare whole-genome coverage uniformity. Analysis of coverage

For Research Use Only. Not for use in diagnostic procedures.

uniformity was performed on a subset of the eight bacterial species:
Pseudomonas aeruginosa (66.2% GC), Escherichia coli (50.8% GC),and Staphylococcus aureus (32.9% GC) (Figure 6).
The coverage plots reveal that the Nextera DNA Flex methods,
including the Flex Direct Colony Method, all achieve greater
uniformity of coverage compared to the Nextera XT Method,
regardless of which organism is tested or its GC content. These
coverage plots also illustrate that the great improvement in coverage
uniformity is not simply a result of adding a greater quantity of DNA to
the library preparations. This is evident from the marked difference incoverage smoothness between the Flex Low-Input Method and the770-2017-036-A | 6

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References

Nextera XT Method; although both methods use the same DNA input(1 ng input), the significantly higher coverage uniformity of the Flex
Low-Input Method reveals that this is a feature intrinsic to the Nextera
DNA Flex chemistry.

1.

Comparison of de novo assembly metrics

2.

Three additional measurements of genome assembly quality were
calculated for comparison. N50 is defined as the minimum
continuous sequence (contig) length needed to cover 50% of the
genome.11 In general, higher N50 values (ie, longer average contigs),
are indicative of better genome assembly. By extension, fewer total
contigs in an assembly is another indicator of high quality, as fewer
long contigs will result in higher accuracy than a high number of small
contigs. For all eight bacterial species, the Nextera DNA Flex libraries
outperformed the Nextera XT libraries for the largest N50s, fewest
number of contigs, and the largest contig sizes (Figure 7). The one
exception was S. agalactiae where similar values, within error, wereobtained for all three assembly metrics.
In all cases, the Nextera DNA Flex libraries, including the quantitationfree Direct Colony libraries, gave identical values, within error, foreach organism for all three metrics (Figure 7). These results
demonstrate the consistency and accuracy of the data generated by
the Nextera DNA Flex Library Prep Kit and the Flex Direct Colony
Method, for various input amounts, and various bacterial species
representing a range of GC content.

Summary
The Nextera DNA Flex Library Preparation Kit offers the fastest, mostflexible library prep protocol in the Illumina portfolio. The On-Bead
Tagmentation chemistry supports a wide range of DNA input
amounts, various sample types, and a broad range of microbiology
applications. The Flex Direct Colony Method has the advantage of
supporting library preparation directly from microbial colonies onculture plates and delivers consistent, highly uniform data from avariety of bacterial species. Furthermore, highly robust data can beobtained without prelibrary prep DNA quantification steps, or
postlibrary prep quantification steps such as library normalization andpooling. See how the innovative Nextera DNA Flex Library Prep
workflow combined with the Flex Direct Colony Method can advanceand accelerate your research goals today.

Reuter S, Ellington MJ, Cartwright EJ, et al. Rapid bacterial whole-genome
sequencing to enhance diagnostic and public health microbiology. JAMA

Intern Med. 2013;173:1397–404.
Quainoo S, Coolen JPM, van Hijum SAFT, et al. Whole-genome sequencingof bacterial pathogens: the future of nosocomial outbreak analysis. Clin

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Rubin BE, Sanders JG, Hampton-Marcell J, et al. DNA extraction protocols
cause differences in 16S rRNA amplicon sequencing efficiency but not incommunity profile composition or structure. MicrobiologyOpen. 2014;3:910–
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van Tongeren SP, Degener JE, Harmsen HJM. Comparison of three rapid
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PCR. Eur J Clin Microbiol Infect Dis. 2011;30:1053–1061.

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Vesty A, Biswas K, Taylor MW, Gear K, Douglas RG. Evaluating the impact ofDNA extraction method on the representation of human oral bacterial andfungal communities. PLoS One. 2017;12:e0169877.

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Illumina (2017). Nextera DNA flex library preparation kit data sheet. Accessed
January 5, 2018.

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NextSeq Control Software and Real-Time Analysis v2 Software.
support.illumina.com/sequencing/sequencing_instruments/nextseq500.html. Accessed January 9, 2018.

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Carver T, Harris SR, Berriman M, Parkhill J and McQuillan JA. Artemis: anintegrated platform for visualization and analysis of high-throughput
sequence-based experimental data. Bioinformatics 2012;28;464–469.

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Bankevich A, Nurk S, Antipov D, et al. SPAdes: A new genome assembly
algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;
19: 455–477.

10. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment toolfor genome assemblies. Bioinformatics. 2013;29:1072–5.
11. Miller JR, Koren S, Sutton G. Assembly algorithms for next-generation
sequencing data. Genomics. 2010;95:315–327.

Learn more
To learn more about the Nextera DNA Flex Library Prep Kit, visit the
Nextera DNA Flex Library Prep page.
For more on microbial genome sequencing with the Nextera DNA
Flex Library Prep Kit, read the Microbial WGS with Nextera DNA Flex
Application Note.
For more on the Flex Direct Colony Method, download the Nextera
DNA Flex Microbial Colony Extraction Protocol

Illumina, Inc. • 1.800.809.4566 toll-free (US) • +1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com© 2018 Illumina, Inc. All rights reserved. All trademarks are the property of Illumina, Inc. or their respective owners. For specific trademark information,
see www.illumina.com/company/legal.html. Pub. No. 770-2017-036-A QB 5650

For Research Use Only. Not for use in diagnostic procedures.

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