Nextera Flex for Enrichment

A fast, integrated workflow for a wide range of target enrichment applications encompassing custom panels, fixed panels, and whole-exome sequencing.

 

Highlights

 

Fast library prep and enrichment workflow

Provides a time-saving solution that is 85% faster than standard Illumina library prep and enrichment

Integrated sample input

Enhances library preparation efficiency with integrated protocols for blood and saliva

Wide range of applications

Enables advanced study designs in cancer research, genetic disease research, and whole-exome sequencing

 

 

Figures and Tables

Figure 1: Nextera Flex for Enrichment assay chemistry—A uniform tagmentation reaction mediated by eBLT followed by a single hybridization reaction enables a fast and flexible workflow.

Parameter

Specification

DNA input type

gDNA, whole blood, saliva, DNA extracted formalin-fixed, paraffin-embedded (FFPE) tissue

DNA input verifieda

10–1000 ng

Sample multiplexing

96 unique dual indexes (UDIs)

Pre-enrichment poolingb

1-plex or 12-plex verified and supported

Supported sequencing systems

All Illumina systems

Total workflow timec

~ 6.5 hours

a. DNA inputs as low as 10 ng are possible, but will not provide saturation-based DNA normalization
b. Other enrichment plexities are possible, but have not been verified. Additional optimization may be required and optimal results are not guaranteed
c. Includes library preparation, enrichment, and library normalization/pooling steps

Table 1: Nextera Flex for Enrichment specifications

Figure 2: Nextera Flex for Enrichment delivers the fastest Illumina enrichment workflow—Workflow times are based on processing 12 samples at 12-plex enrichment. Times may vary depending on equipment used, number of samples processed, automation procedures, or user experience. Nextera Flex for Enrichment is up to 85% faster than standard Illumina library prep and enrichment workflowshis is the figure caption.

 

Nextera Flex for Enrichment

TruSeq DNA Exome

Nextera DNA Exome

Nextera Rapid Capture Custom Enrichment

Integrated DNA lysis optiona

ü

Flexible, broad DNA input range

ü

Library normalization includedb

ü

DNA input required

≥ 50 ng

100 ng

50 ng

50 ng

Sample type

DNA and FFPE

DNA and FFPE

DNA

DNA

Total library prep and enrichment timec

~6.5 hours

2.5 days

2 days

1.5 days

Insert sized

150–220 bp

150 bp

150–220 bp

230 bp

Sample index setse

96 unique dual indexes

24 single indexes, 96 dual indexes

24 single indexes, 96 dual indexes

24 single indexes,
96 dual indexes

a. Integrated lysis protocols available for blood and saliva
b. Library normalization occurs with ≥ 50 ng gDNA input
c. Total library prep and enrichment time includes library preparation, enrichment, and library normalization/pooling steps
d. Degraded FFPE DNA may result in smaller insert sizes
e. Pre-enrichment pooling of up to 12-plex has been verified and is supported

Table 2: Comparison of Illumina enrichment workflows

Figure 3: Nextera Flex for Enrichment provides optimized performance across Illumina sequencing systems—The Nextera Flex for Enrichment solution is compatible with all Illumina sequencing systems, including high-throughput, production-scale systems.

Parameterb

Nextera Flex for Enrichment

Nextera Flex for Enrichment

Nextera Flex for Enrichment

TruSeq DNA Exome

Nextera DNA Exome

Panel

Illumina Exome Panel

Exome Panel X

Exome Panel Y

Illumina Exome Panel

Illumina Exome Panel

Panel size

45 Mb

39 Mb

33 Mb

45 Mb

45 Mb

Probe size

80 bp

120 bp

120 bp

80 bp

80 bp

Padded read enrichment    (on-target)

85%

91%

91%

85%

> 75%

Fragment length median

~200 bp

~200 bp

~200 bp

~150 bp

150–250 bp

Coverage at 20´

93%

96%

97%

> 90%

> 85%

Uniformity of coverage

95%

97%

98%

Read depth per sample

30M PF clusters

25M PF clusters

20M PF clusters

40M PF clusters

40M PF clusters

SNV precision

99%

99%

99%

99%

99%

SNV recall

94%

94%

95%

89%

91%

a. Data represents example comparison data. Actual performance specifications may vary.
b. The analysis was run on 48 samples (all NA12878 Coriell samples) per condition. Data analysis was performed using the Enrichment BaseSpace App.

 

 

 

Table 3: Comparison of performance

Panel

iSeq 100 System

MiniSeq System

MiSeq System

NextSeq Series

Panel

NextSeq Series

HiSeq Series

NovaSeq 6000 System

 

Mid

High

v2

v2 Nano/Micro

v3

Mid

High

Mid

High

2500 RR/HO

3000/4000

SP

S1

S2

S4

Fixed panels

Exome Panel

TruSight One

NR

NR

2

1

0/0

2

12

36

Illumina Exome

5

16

12/80

100

64

128

164

384

TruSight One Expanded

NR

NR

1

0

0/0

1

7

24

Exome Panel X

5

16

12/80

100

64

128

164

384

TruSight Cancer

4

8

24

12

1/4

24

96

384

Exome Panel Y

6

20

15/100

125

80

160

205

500

TruSight Hereditary Cancer

3

5

16

8

1/2

16

80

250

 

 

 

 

       

 

TruSight Cardio

4

8

24

12

1/4

24

96

384

 

 

 

 

       

 

Custom panels

2000 probes

8

16

48

30

2/8

48

260

384

 

 

 

 

       

 

5000 probes

2

4

12

8

1/2

12

65

200

 

 

 

 

       

 

10,000 probes

1

2

6

4

0/1

6

33

100

 

 

 

 

       

 

Abbreviations: Mid, mid output; High, high output; RR, rapid-run mode; HO, high-output mode (v4)

Table 4: Sample throughput per flow cell with Nextera Flex for Enrichment

Figure 4: High coverage uniformity and padded read enrichment—Nextera Flex for Enrichment provides high coverage uniformity and on-target padded read enrichment for custom, fixed, and exome panels.

Figure 5: Accurate variant calling—Nextera Flex for Enrichment provides low abundance somatic variant calling for (A, B) cell line FFPE control human reference DNA samples and (C, D) real world FFPE samples with observed variant frequency showing significant correlation with expected frequencies.

Figure 6: Broad range of applications with Nextera Flex for EnrichmentNextera Flex for Enrichment can be used to support a broad range of applications including fixed panels, custom panels, and whole-exome sequencing.

 

Introduction

The Nextera Flex for Enrichment solution combines versatile, simple, and fast library prep and enrichment functionality for targeted enrichment and exome sequencing applications. It offers extraordinary flexibility for input type, input amount, and a wide range of supported enrichment sequencing applications encompassing custom panels, fixed panels, and whole-exome sequencing (Table 1).

Nextera Flex for Enrichment uses innovative bead-based chemistry with a simplified, single hybridization step (Figure 1). With the Nextera Flex for Enrichment workflow, DNA extraction can be processed directly from fresh blood and saliva samples with the Flex Lysis Reagent Kit and Saliva Lysis Protocol, respectively, for additional time savings.

Fast and flexible library preparation and enrichment workflow

A key component of the Nextera Flex for Enrichment solution is On-Bead Tagmentation, which uses bead-bound transposomes to mediate a uniform tagmentation reaction. This strategy provides several significant advantages.

  • For gDNA inputs ≥ 50 ng, accurate quantitation of the initial DNA sample is not required, as insert fragment size is not affected, saving time and costs associated with kits and reagents.

  • On-Bead Tagmentation eliminates the need for separate DNA fragmentation steps, saving time and costs associated with related consumables.

  • For gDNA inputs between 50–1000 ng, saturation-based DNA normalization eliminates the need for individual library quantitation and normalization steps before enrichment.

  • Novel 90-minute single hybridization protocol enables enrichment in less than four hours.

Fastest Illumina enrichment workflow

The Nextera Flex for Enrichment solution supports liquid-handling systems for library prep automation and produces a workflow with the lowest number of steps and the fastest total workflow time in the Illumina portfolio (Figure 2)(Table 2).

Integrated DNA Input

DNA extraction can be processed directly from whole blood or saliva samples. The optional Flex Lysis Reagent Kit has been optimized and validated for Nextera Flex for Enrichment library preparation from whole blood and is integrated into the workflow for maximum efficiency. The lysis protocols feature bead-based reagents and require less than 30 minutes of hands-on time.

Optimized performance across Illumina sequencing systems

The robust and straightforward Nextera Flex for Enrichment solution yields reliable results across all Illumina sequencing systems by providing > 90% on-target reads, > 95% uniformity, and low PCR duplicate rate (Table 3). Nextera Flex for Enrichment is optimized for high-throughput systems (Figure 3)(Table 4).

DNA enrichment for a broad range of applications

By combining exceptional enrichment performance and the proven accuracy of Illumina sequencing by synthesis (SBS) chemistry,1 the Nextera Flex for Enrichment solution supports both fixed and custom panels of varying sizes, including whole-exome sequencing, for customers who need advanced study designs in a variety of areas (Figure 6). Furthermore, Nextera Flex for Enrichment is compatible with Illumina and third-party enrichment probes/panels, which enables content portability for increased flexibility.

Accurate Data

Nextera Flex for Enrichment produces highly uniform and consistent insert sizes, across a wide DNA input range, delivering uniform and consistent library yields.2 In addition, Nextera Flex for Enrichment provides high coverage uniformity and padded read enrichment for custom, fixed, and exome panels (Figure 4). Also, Nextera Flex for Enrichment enables accurate single nucleotide variant (SNV) and insertion/deletion (indel) recall and precision, as compared to other Illumina enrichment solutions (Figure 5 and Table 3).

Summary

Nextera Flex for Enrichment features the fastest workflow in the Illumina enrichment portfolio. The user-friendly, automation-compatible solution supports users of all experience levels and provides a common workflow for a variety of experimental designs. On-Bead Tagmentation chemistry enables support for a wide range of DNA input amounts, various sample types, and a broad range of applications, including fixed panels, custom panels, and whole-exome sequencing. Furthermore, Nextera Flex for Enrichment is compatible with Illumina and third-party enrichment probes/panels, which enables content portability. The innovative Nextera Flex for Enrichment solution combined with the power of Illumina SBS chemistry provides an optimal targeted enrichment and exome sequencing experience.

Ordering Information

Product

Catalog No.

Nextera DNA Flex Pre-Enrichment Library Prep and Enrichment Reagents
16 samples (16, 1-plex enrichment reactions)

20025523

Nextera DNA Flex Pre-Enrichment Library Prep and Enrichment Reagents
96 samples (8, 12-plex enrichment reactions)

20025524

Nextera DNA Flex Pre-Enrichment Library Prep Reagents (16 samples)

20025519

Nextera DNA Flex Pre-Enrichment Library Prep Reagents (96 samples)

20025520

Flex Lysis Reagent Kit (for blood lysis)

20018706

IDT for Illumina - Nextera DNA Unique Dual Indexes - Set A (96 indexes, 96 samples)

20027213

Illumina Exome Panel (8 enrichment reactions)

20020183

TruSight One (6 enrichment reactions)

20029227

TruSight One Expanded (6 enrichment reactions)

20029226

TruSight Cancer (8 enrichment reactions)

FC-121-0202

TruSight Hereditary Cancer (8 enrichment reactions)

20029551

TruSight Cardio (1 enrichment reaction)

20029228

TruSight Cardio (8 enrichment reactions)

20029229

TruSeq Neurodegeneration (8 enrichment reactions)

20029550

Illumina Custom Enrichment Panel

20025371

References

1. Bentley DR, Balasubramanian S, Swerdlow HP, et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 2008;456:53-59.
2. Illumina (2017). Nextera DNA Flex Library Preparation Kit Data Sheet. Accessed September 10, 2018.

 

Download Data Sheet PDF

 

 

 

Additional Resources

Library Prep and Array Kit Selector

This tool will help you determine the best kit for your needs based on your project type, starting material, and the sequencing method or application.

 

Learn more

Custom Protocol Selector

Custom Protocol Selector is now faster, mobile-friendly, and even more flexible. Customize your level of detail, add notes, and print directly from the browser.

 

Learn more

Illumina Concierge Services

Access a range of support options including advanced content design, assistance assessing custom enrichment, in-person training, and more.

 

Learn more

 

 

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© 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. 770-2018-002-A  QB 6494