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Nextera™ crude lysate protocol for
metagenomic next-generation sequencing
The Nextera DNA Flex Library Prep Kit with the Nextera Crude Lysate Protocol deliver fast,accurate metagenomic profiling and high-quality de novo genome assembly data.

Introduction
Next-generation sequencing (NGS) technology has become an
important tool in metagenomics research for organism identification,
12
metagenomic profiling, and de novo assembly. , The Nextera library
prep portfolio introduced several workflow advantages that support
faster and more efficient library preparation for metagenomic
applications (Figure 1). Tagmentation chemistry and the release of
Nextera DNA and Nextera XT DNA Library Preparation Kits
consolidated DNA fragmentation and adapter tagging steps into asingle reaction.3 With innovative on-bead tagmentation, the Nextera
DNA Flex Library Prep Kit further shortened the library prep workflow
by integrating DNA extraction, quantitation, fragmentation, and librarynormalization steps.4
Though NGS-based whole-genome sequencing (WGS) has provided
significant advantages in speed, accuracy, and depth of information to
microbiology labs, DNA extraction and library preparation steps haveremained a significant bottleneck in the NGS workflow. Preparation of
NGS libraries for metagenomic studies typically begins with timeconsuming and labor-intensive genomic DNA extraction steps. To
address this challenge in metagenomics, Illumina offers the Nextera
DNA Flex Library Preparation Kit with the Nextera Crude Lysate
Protocol—a library prep method that supports quick and easy library
preparation directly from crude lysate. Sequencing directly from crude
lysate eliminates the time and cost associated with DNA extraction
steps. In addition to increased speed and efficiency, the Nextera DNA
Flex Library Preparation Kit offers exceptional flexibility for sample input
type and cell lysis methods, including direct bacterial colonies, blood,and saliva.
This application note demonstrates the performance of the Nextera
DNA Flex Library Prep Kit using the Nextera Crude Lysate Protocol
using mock microbial communities and real-world human stool
samples. This application note also shows how the Nextera DNA Flex
Library Prep Kit, with its advanced chemistry and faster workflow,
outperforms the original Nextera DNA Library Prep Kit in metagenomic
profiling, and de novo assembly.*

Figure 1: Nextera Crude Lysate Protocol for Metagenomics—The Nextera DNA
Flex Kit with the Nextera Crude Lysate Protocol enables library prep directly fromcrude lysate, resulting in additional time and cost savings while retaining dataconsistency and quality.

Methods
Sample sources
To represent mock microbial communities, 20 Strain Even Mix
Genomic Material (ATCC MSA-1002), 20 Strain Staggered Mix
Genomic Material (ATCC MSA-1003), and 20 Strain Even Mix Whole
Cell Material (ATCC MSA-2002) were used. The 20 Strain
Even/Staggered Mix Genomic Material mixtures are composed of
purified DNA extracted from 20 bacterial strains, while the 20 Strain
Even Mix Whole Cell Material is a mixture of whole (unlysed) cells. The
species in these mixtures were selected for their diverse genome size,
range of GC content, and Gram stain profile. Additionally, themicroorganisms in the 20 strain mixtures have fully sequenced andcharacterized genomes. To represent real-world metagenomic
samples, stool samples were obtained from both healthy donors andpatients undergoing drug treatment.

*The original Nextera DNA Library Prep and Nextera XT DNA Library Prep Kits use tagmentation chemistry, while the Nextera DNA Flex Library Prep Kits use
advanced on-bead tagmentation chemistry. On-bead tagmentation chemistry delivers more uniform library yield, consistent insert sizes, and uniform genome
coverage compared to the original tagmentation chemistry.

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

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Crude lysate and DNA extraction from mock microbialcommunities
To prepare crude lysate, 200 µl from the 20 Strain Even Mix Whole Cell
sample were processed as described in the Nextera Crude Lysate
Protocol with the PureLink Microbiome DNA Purification Kit (Catalog
No. A29790, ThermoFisher). Supernatant from step H, which includes
the transfer of supernatant from homogenized cells to a clean tube,
was used as crude lysate.5 To prepare extracted DNA, an equivalent
number of cells were processed with the PureLink Microbiome DNA
Purification Kit using the full protocol. The Nextera Crude Lysate
Protocol is compatible with a variety of bacterial DNA extraction kits
(Table 1).

Crude lysate and DNA extraction from stool samples
Stool samples from donor patients were used to generate crude lysatesand extracted DNA as described in the Nextera Crude Lysate Protocol
from Stool Samples with the PureLink Microbiome DNA Purification Kit.
0.05 g of stool samples were processed with the PureLink Microbiome
DNA Purification Kit. Supernatant from step G, which includes thetransfer of supernatant from homogenized cells to a clean tube, wasused as crude lysate.6 To prepare extracted DNA, an equivalentamount of starting material was processed with the PureLink
Microbiome DNA Purification Kit using the full protocol. Stool samples
can be highly inhibitory due to the presence of PCR inhibitors such as
bile, salts, and polysaccharides. Inhibited library preps show an inability
to pellet during clean-up steps or they may deliver low library yield.
Decreasing the amount of lysate below the recommended amount
may be needed for some highly inhibitory samples. The Nextera Crude
Lysate Protocol is compatible with a variety of stool sample purification
kits (Table 1).

To compare the performance of crude lysate to extracted DNA,
sequencing libraries were prepared with 5 µl lysate or 5 µl extracted
DNA from the 20 Strain Even Mix Whole Cell sample or from stool
samples. The lysate and extracted DNA samples were used with the
Nextera DNA Flex Library Preparation Kit using either the Nextera
Crude Lysate Protocol or the standard protocol. Library preparation
using the Nextera Crude Lysate Protocol significantly reduced thetotal amount of time and the number of touch points in the library
preparation workflow (Figure 2). By using crude lysate as a direct input
into the Nextera DNA Flex Library Preparation Kit, the need for costly,
time-consuming DNA quantitation kits are also eliminated.
For a comparison of the Nextera DNA Flex Library Preparation Kit andthe original Nextera DNA Library Preparation Kit (Catalog No. FC121-1030, Illumina), 50 ng DNA from the 20 Strain Even Mix Genomic
Material sample and 50 ng DNA from the 20 Strain Staggered Mix
Genomic Material sample were added directly to the library prep kits.
Libraries were prepared using the standard protocol.
All libraries were sequenced on the NextSeq™ 550 Sequencing
System with a run configuration of 2 × 150 bp paired-end reads using
the NextSeq 500/550 High Output v2 Kit (Catalog No. FC-404-2004,
Illumina).

Table 1: DNA extraction and lysate prep kits aKits for bacterial samples
PureLink Microbiome DNA Purification Kit (ThermoFisher)
UltraClean Microbial DNA Isolation Kit (MOBIO)
ChargeSwitch gDNA Mini Bacteria Kit (ThermoFisher)
Kits for stool samples
PureLink Microbiome DNA purification kit (Invitrogen)
PowerSoil DNA Isolation Kit (MOBIO)
PowerFecal DNA Isolation Kit (MOBIO)
QIAamp DNA Stool mini kit(Qiagen)a. For detailed information about recommended collection and lysis methods
to obtain crude lysate with these kits, and the recommended input amountof crude lysate, download the Nextera Crude Lysate Protocol Guide.

Library preparation and sequencing
To test the accuracy and sensitivity of the Nextera DNA Flex Library
Prep Kit with species identification and metagenomic profiling, 10 ng
of DNA from the 20 Strain Staggered Mix Genomic Material sample
was added directly to the Nextera DNA Flex Library Prep Kit (Catalog
No. 20018704, Illumina) and prepared using the standard protocol(extracted DNA).

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

Figure 2: Comparison of extracted DNA and crude lysate workflows—The Nextera
Crude Lysate Protocol reduces the amount of time and total number of touchpoints required in the library preparation protocol. The crude lysate protocol also
reduces the time and cost associated with additional purification and quantitation
steps. Workflow times were calculated assuming specific methods: DNA
extraction (PureLink Microbiome DNA Purification Kit), DNA Quantitation (Qubit).
Times may vary depending on equipment used, kits used, sample batch number,
automation procedures, and user experience.

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Data analysis
Metagenomic profiling metrics, including true positives, relative
abundance, false positives, observed abundance, and expected
abundance, were calculated on the One Codex7 platform. Genome
assembly bar graphs were generated with two software tools. FirstMEGAHIT v1.1.1.8 performed de novo alignment with the FASTQ
reads to generate contigs, then the contigs were assessed with
Quality Assessment Tool for Genome Assemblies (QUAST) 9 with the
known reference genomes. Metagenomic profiling stacked bar
graphs were compiled with GENIUS Metagenomics software.10
GENIUS Metagenomics software can be accessed in BaseSpace™
Sequence Hub, the Illumina genomics computing platform.

Table 3: 20 Strain Staggered Mix metagenomic profilingcomposition statistics
Observed
abundance

Expected
abundance

Rhodobacter sphaeroides

19.07%

18.00%

Escherichia coli

18.54%

18.00%

Staphylococcus epidermidis

18.26%

18.00%

Porphyromonas gingivalis

17.17%

18.00%

Streptococcus mutans

17.61%

18.00%

Pseudomonas aeruginosa

1.93%

1.80%

Clostridium beijerinckii

1.80%

1.80%

Bacillus cereus

1.15%

1.80%

Staphylococcus aureus

1.64%

1.80%

Streptococcus agalactiae

1.77%

1.80%

Results

Acinetobacter baumannii

0.18%

0.18%

Propionibacterium acnes

0.21%

0.18%

Metagenomic profiling with mock community mixtures andstool samples

Neisseria meningitidis

0.21%

0.18%

Lactobacillus gasseri

0.19%

0.18%

Helicobacter pylori

0.19%

0.18%

Bacteroides vulgatus

0.015%

0.018%

Enterococcus faecalis

0.021%

0.018%

Deinococcus radiodurans

0.017%

0.018%

Bifidobacterium adolescentis

0.013%

0.018%

Actinomyces odontolyticus

0.007%

0.018%Name of organism

To establish the accuracy and sensitivity of the Nextera DNA Flex
Library Prep Kit in basic organism identification, a sequencing library
was prepared with the 20 Strain Staggered Mix sample.
Metagenomic profiling summary metrics—true positive, relative
abundance, and false positives—were calculated on the One Codex
platform. The results demonstrate excellent metagenomic profilingperformance: all 20 organisms in the 20 Strain Staggered Mix wereidentified with no false positives (Table 2). The composition statistics
with the 20 Strain Staggered Mix were also highly accurate with theobserved abundance scores closely matching the expected
abundance scores for all 20 species (Table 3). Furthermore, theobserved abundance scores spanned four orders of magnitude andwere able to detect species present in the sample down to 0.018%,
which demonstrates high sensitivity in the Nextera DNA Flexsequencing workflow.

The Nextera DNA Flex Crude Lysate Protocol vs. thestandard protocol with mock microbial communities

Table 2: 20 Strain Staggered Mix metagenomic profilingsummary statistics a,b

Table 4: Comparison of crude lysate and extracted DNA
metagenomic profiling summary data

Library
20 Strain
Staggered Mix
and Nextera DNA
Flex Library Prep
Kit

True positives

100%

Relative
abundance

100%

False positives

0a. One Codex defines statistics as follows: True Positives: The percentage oforganisms present in the control. Organisms are marked as "Present" if they
are detected within two logs of the true abundance. Relative Abundance:
The Pearson correlation coefficient between the known input organism
abundances and the detected abundances (based on genome-size adjusted
read counts). False Positives: 100% less 10 percentage points for each"High" abundance false positive, 5 points for each "Moderate" one, and 1
point for each "Low" one. "Trace" false positives do not count against thescore, and the minimum possible score is 0%.
b. Data set down sampled from 20 million reads to 1 million reads.

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

To compare the performance of the Nextera Crude Lysate Protocol
to the standard protocol, sequencing libraries were prepared with
crude lysate and extracted DNA from the 20 Strain Even Mix Whole
Cell Material sample. Libraries made with crude lysate and extracted
DNA generated equivalent metagenomic profiling summary statistics(Table 4).

True

False

positives

Relative
abundance

positives

Crude Lysate

100%

33%

0

Extracted
DNA

100%

32%

0

Libraries

a. Data set down sampled from 20 million reads to 5 million reads.

Using the same crude lysate and extracted DNA data set, the de novo
genome assembly quality was also compared and the percentage ofgenome fraction assembled was calculated with QUAST. In general,a higher fraction of genome assembled is indicative of a higherquality genome assembly. For all 20 organisms analyzed, the crude
lysate and extracted DNA libraries generated very similar genome
assembly results (Figure 3).

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Figure 3: Comparison of crude lysate and extracted DNA genome assembly—Assembly was performed with MEGAHIT using 5 million, paired-end, 150 bp reads. Bargraph illustrates genome assembly performance for 20 organisms as reported by QUAST.

Figure 4: Comparison of crude lysate and extracted DNA metagenomic profiles—–Bar graph illustrates the abundance of each organism identified by GENIUS
metagenomics analysis (using 3 million, paired-end, 150 bp reads) for the > 60 organisms identified in both stool libraries.

The Nextera DNA Flex crude lysate protocol vs. thestandard protocol with stool samples
The metagenomic profiles resulting from the Nextera Crude Lysate
Protocol and the standard Nextera DNA Flex Library Prep protocol arevery similar even with highly inhibitory, challenging stool samples(Figure 4). Unlike the mock microbial community samples, the stool
samples contain a mixture of unknown species and an unknown
species composition (percentage of each species). For the de novo
assembly of metagenomics samples with unknown species,
cumulative assembled length can be used to assess the quality ofgenome assembly. Assembly using QUAST revealed that the
cumulative assembled lengths are highly similar between libraries
prepared with the Nextera Crude Lysate Protocol from Stool Samples
and the standard Nextera DNA Flex Library Prep protocol (Figure 5).
Figure 5: Comparison of crude lysate and extracted DNA genome assembly withunknown species—Graph illustrates cumulative assembled length (Mbp) with thecontigs ordered from largest to smallest. 20 million reads were used for assembly.

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

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Figure 6: Comparison of Nextera DNA Flex and original Nextera genome assembly metrics—Bar graph illustrates genome fraction assembled with MEGAHIT (using 2
million, paired-end, 150 bp reads) for all 20 organisms. Organisms are listed in the order of increasing GC% from left to right.

Figure 7: Comparison of Nextera DNA Flex and original Nextera DNA metagenomic profiles—Bar graph illustrates the abundance of each organism identified by GENIUS
metagenomics analysis (3 million, paired-end, 150 bp reads were used for analysis) for the > 60 organisms identified in both libraries.

Nextera DNA Flex outperforms the original Nextera DNA
Library Prep Kit for metagenomic profiling

Nextera DNA Flex enables superior genome assemblycompared to the original Nextera DNA Library Prep Kit

The profiling performance of the Nextera DNA Flex Library Prep Kit
was compared with the original Nextera DNA Library Prep Kit withlibraries prepared from the 20 Strain Even Mix and the 20 Strain
Staggered Mix Genomic Material samples. Summary metrics were
calculated on the One Codex platform and the results demonstratethat the Nextera DNA Flex Library Prep Kit delivers superior
metagenomic profiling results compared to the original Nextera DNA
Library Prep Kit (Table 5). Unlike Nextera DNA and Nextera XT Library
Prep Kits, Nextera DNA Flex Library Prep Kits use advanced on-beadtagmentation, which delivers uniform library yield, consistent insert
sizes, and uniform genome coverage. These advantages work
together to produce improved metagenomic profiling.

Beyond assessments of organism detection and metagenomic
profiling, the sequencing data were analyzed to compare genome
assembly metrics. To evaluate genome assembly quality, thegenome fraction assembly metrics were calculated by QUAST. The
QUAST data show that Nextera DNA Flex outperformed the original
Nextera DNA Kit for most of the 20 organisms in the 20 Strain Even
Mix Whole Cell Material sample, especially for the AT rich organisms(Figure 6).

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

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Table 5: Comparison of Nextera DNA Flex Library Prep and theoriginal Nextera DNA Library Prep metagenomic profilingsummary data
True
positives

Relative
abundance

False
positives

20 Strain Even Mix Genomic Material

Ordering Information
Product

Catalog No.

Nextera DNA Flex Library Prep Kit(24 samples)

20018704

Nextera DNA Flex Library Prep Kit(96 samples)

20018705

Nextera DNA Flex Library Prep Kit(50 ng)Flex Lysis Reagent Kit

20018706

100%

97%

0

20018707

Nextera DNA Library Prep Kit(50 ng)Nextera DNA CD Indexes(24 indexes, 24 samples)

100%

83%

0

Nextera DNA CD Indexes(96 indexes, 96 samples)

20018708

20 Strain Staggered Mix Genomic Material
Nextera DNA Flex Library Prep Kit(50 ng)

100%

100%

0

Nextera DNA Library Prep Kit(50 ng)

100%

96%

0

CD Indexes: Combinatorial Dual Indexes. 24 dual indexes provided to support
up to 24 samples or 96 dual indexes provided to support up to 96 samples.
Single Indexes: 24 single indexes provided to support up to 96 samples.

References
To test the performance of the Nextera DNA Flex Library Prep Kit
compared to the (original) Nextera DNA Library Prep Kit with realworld metagenomic samples, crude lysate and extracted DNA
libraries were prepared from stool samples. The metagenomics
profiles resulting from the two library prep kits are very similar,
indicating that both provide excellent and comparable profile results(Figure 7).

Summary
The Nextera DNA Flex Library Prep Kit with the Nextera Crude Lysate
Protocol provide a fast and easy workflow that eliminates costly, timeconsuming DNA extraction steps and delivers exceptional data
quality. The Nextera Crude Lysate Protocol offers high sensitivity andaccuracy for organism detection and metagenomic profiling, as wellas high-quality de novo genome assembly comparable to extracted
DNA. With the ability to support complex metagenomic mixtures suchas stool or mock microbial community samples with the Nextera
Crude Lysate Protocol, as well as saliva, blood, and direct bacterialcolonies with additional demonstrated protocols, the Nextera DNA
Flex Library Prep Kit delivers a flexible, cost-effective approach for
NGS-based metagenomic research.

1.

Ranjan R, Asha RA, Metwally A, McGee HS, Perkins DL. Analysis of the
microbiome: Advantages of whole genome shotgun versus 16S ampliconsequencing. Biochem Biophys Res Commun. 2016;469:967–977.

2.

Lloyd-Price J, Mahurkar A, Rahnavard G, et al. Strains, functions anddynamics in the expanded Human Microbiome Project. Nature.
2017;550:61-66.

3.

Illumina (2016). Nextera DNA Library Preparation Kit Data Sheet. Accessed
April 10, 2018.

4.

Illumina (2017). Nextera DNA Flex Library Preparation Kit Data Sheet.
Accessed April 10, 2018.

5.

Invitrogen (2015). PureLink Microbiome DNA Purification Kit User Guide forMicrobial Culture. Accessed April 12, 2018.

6.

Invitrogen (2015). PureLink Microbiome DNA Purification Kit User Guide for
Stool Samples. Accessed April 12, 2018.

7.

One Codex platform. www.onecodex.com. Accessed April 12, 2018.

8.

MEGAHIT v1.1.1. github.com/voutcn/MEGAHIT. Accessed April 12, 2018.

9.

QUAST. quast.sourceforge.net/quast.html. Accessed April 13, 2018.

10. GENIUS Metagenomics. www.illumina.com/products/by-type/informaticsproducts/basespace-sequence-hub/apps/cosmosid-genius-metagenomicsknow-now.html. Accessed April 12, 2018.

Learn more
To learn more about the Nextera DNA Flex Library Prep Kit, visit
www.illumina.com/nextera-dna-flex
For more on direct bacterial colony sequencing with Nextera DNA
Flex, download www.illumina.com/content/dam/illuminamarketing/documents/products/appnotes/nextera-dna-flex-directcolony-app-note-770-2017-036.pdf
For more on microbial genome sequencing with the Nextera DNA
Flex, visit www.illumina.com/content/dam/illuminamarketing/documents/products/appnotes/nextera-dna-flex-smallgenomes-application-note-770-2017-019.pdf

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. 770-2018-006-A QB 5996

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

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