HyperCap Workflow v2.0Integrating KAPA library preparation and SeqCap target enrichment

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

The HyperCap Workflow v2.0 accelerates DNA target enrichment by combining two industry-leading solutions into an integrated, automatable workflow. Libraries generated with KAPA library preparation kits provide the foundation for Roche SeqCap target enrichment, producing high-quality sequencing-ready libraries in less than two days.

• KAPA Dual-Indexed Adapters enable greater multiplexing, maximizing sequencing resources

• NEW Roche Universal Blocking Oligos (UBOs) reduce workflow complexity and hands-on time by eliminating the need for adapter-matched blocking oligos

• Flexible input options enhance success with low-input and poor-quality samples

• Validated automation scripts on numerous platforms enable increased throughput

• Partnering with a single, trusted vendor saves time on ordering, service, and support

STREAMLINEDWORKFLOW

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

Figure 1. The HyperCap v2.0 streamlined two-day workflow for Illumina® sequencing. This workflow is compatible with both the KAPA HyperPrep Kit, with mechanically-fragmented input DNA, and the KAPA HyperPlus Kit, which includes enzymatic fragmentation.

With KAPA Dual-Indexed Adapters enabling greater multiplexing and maximizing sequencing resources

SeqC

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Adapter Ligation

Input DNA

Fragmentation

End Repair and A-tailing

Bead Cleanup andSize Selection

Hybridization

Pre-Capture Amplification and Quantification

Sequencing

Pooling(Optional)

Capture Bead Binding and Wash

Amplification and Quantification

Sequencing-readyDNA in <2 days

With NEW Roche UBOs reducing workflow complexity and hands-on time by eliminating the need for adapter-matched blocking oligos

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3Data on file. For Research Use Only. Not for use in diagnostic procedures.

Increase convenience with Roche Universal Blocking Oligos (UBOs)• Use a single tube of oligos to efficiently block barcoded adapters

• Select single- or dual-indexing strategies

• Meet or exceed the performance of Supplier X’s blocking oligos while maintaining uniformity of capture

Figure 2. Performance metrics were compared between experiments using Roche UBOs, Roche HE-Oligos, and Supplier X’s blocking oligos. Target-enriched libraries were prepared from 100 ng of HapMap NA12891 DNA using the HyperCap Workflow v2.0. The KAPA HyperPrep Kit and the indicated adapters were used to create the libraries, and target enrichment was performed with the SeqCap EZ Inherited Disease Panel and the indicated blocking oligos. The combined use of KAPA Dual-Indexed Adapters and Roche UBOs resulted in equivalent-to-better performance with respect to both the on-target rate and the Fold 80 base penalty (a measure of sequencing depth uniformity, for which lower #s are better). Error bars represent the standard deviation across triplicate samples. For this and all subsequent data, sequencing was performed using an Illumina® HiSeq® 4000 instrument with 2 x 150 read length. Reads were randomly subsampled to 12.3 M for comparative analyses.

Figure 3. Target-enriched libraries were prepared from 100, 50, or 10 ng of HapMap NA12891 DNA using the HyperCap Workflow v2.0. The KAPA HyperPrep Kit and KAPA Dual-indexed Adapters were used to create the libraries, and target enrichment was performed with the SeqCap EZ Inherited Disease Panel and Roche UBOs. For the 50 and 10 ng inputs, size selection was omitted to preserve sample complexity. Although these low-input samples yielded high-quality data, there was a small decrease in on-target rates, which is to be expected with size selection omitted. Data from low-input samples also showed an increase in duplication rates, as well as reduced base coverage at greater sequencing depths. Error bars, when present, represent the standard deviation across triplicate samples. The 10 ng bars represent data for a single library.

Address low-input sample needs• Achieve high coverage across a range of sample inputs

• Reduce duplicate rates with industry-leading conversion efficiency

On-

targ

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, %

50

25

0

75

100

Roche UBOs/DI Adapters

80

Roche HE-Oligos/SI Adapters

75

Supplier X UBOs/Roche DI Adapters

78

Fold

80

base

pen

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1.50

1.25

1.00

1.75

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Roche UBOs/DI Adapters

1.71

Roche HE-Oligos/SI Adapters

1.72

Supplier X UBOs/Roche DI Adapters

1.71

Duplicate reads

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On-target reads Bases covered >20X Bases covered >40X

100 ng 50 ng 10 ng

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9 8

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73 73

98 97 96

8780

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Data on file.For Research Use Only. Not for use in diagnostic procedures.HYPERCAP, KAPA, and SEQCAP are trademarks of Roche. All other product names and trademarks are the property of their respective owners. © 2018 Roche Sequencing and Life Science. All rights reserved. SS507001 A337 1/18

Published by:

Roche Sequencing and Life Science 9115 Hague Road Indianapolis, IN 46256

sequencing.roche.com/hypercap

Ordering information for HyperCap Workflow v2.0Streamline purchasing and support for all your library prep and target enrichment needs from a single trusted vendor.

Workflow Step Product Name Pack Size Catalog #

Library Prep KAPA HyperPrep Kit with KAPA Library Amplification Primer Mix (10X) for mechanically fragmented DNA

8 reactions KK850024 reactions KK850296 reactions KK8504

KAPA HyperPlus Kit with Library Amplification with enzymatic fragmentation

8 reactions KK851024 reactions KK851296 reactions KK8514

Enrichment Reagents

HyperCap Target Enrichment Kit Reagents for hybridization, wash, and amplification steps, including Roche Universal Blocking Oligos

24 reactions 0828637000196 reactions 08286345001

Adapters SeqCap Adapter Kit A Single-indexed adapters for multiplexed capture and sequencing, validated for HyperCap Workflow v2.0

96 reactions 07141530001

SeqCap Adapter Kit B Single-indexed adapters for multiplexed capture and sequencing, validated for HyperCap Workflow v2.0

96 reactions 07141548001

KAPA Dual-Indexed (96) Adapters Barcoded adapters for multiplexed sequencing, validated for HyperCap Workflow v2.0

96 Adapters x 20 µL KK8722

Beads HyperCap Bead Kit Capture and selection/purification beads

24 reactions 0828641800196 reactions 08286400001

Probes SeqCap EZ Probes Shared Designs (validated fixed content) or custom-designed EZ probe pools; sequencing.roche.com

4 – 960 reactions Assorted

Also Available Separately

Blocking Oligos Roche Universal Blocking Oligos Designed for blocking dual- or single-index barcoded adapters

24 reactions 0828639600196 reactions 08286388001

Generate high-quality libraries from degraded samples• Elevate the quality of FFPE sequencing data

• Reduce the impact of DNA quality on sequencing depth

Figure 4. Target-enriched libraries were prepared from 25 ng of DNA from a paired set of frozen/FFPE liver samples using the HyperCap Workflow v2.0. The KAPA HyperPrep Kit and KAPA Dual-indexed Adapters were used to create the libraries, and target enrichment was performed with the SeqCap EZ Inherited Disease Panel and Roche UBOs; size selection was omitted. Prior to library preparation, input DNA quality was assessed via the KAPA hgDNA Quantification and QC Kit, and Q-ratios were determined to be 0.9 and 0.7 for frozen and FFPE samples, respectively. Although a slight increase in duplicate reads was observed for libraries made from FFPE samples, other key metrics such as on-target reads and base coverage were not impacted. Error bars represent the standard deviation across triplicate samples.

Perc

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Frozen

FFPE

0

25

50

75

100

7978

9797

7273

1511

Duplicate reads On-target reads Bases covered >20X Bases covered >40X