Application Highlight:Human Whole-Genome Sequencing Workflow

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

ROCHE SEQUENCINGSEQ SUCCESS WITH EVERY SAMPLE

Every sample is precious: Seq success with the fully integrated Roche Sequencing NGS WorkflowWhole-genome sequencing (WGS) provides access to the wealth of information inside every human DNA sample. Roche offers complete sequencing workflows to unlock this potential—from automated DNA extraction through accurate library quantification—to help you achieve success in sequencing on the Illumina® HiSeq® and NovaSeq™ platforms.

From sample to sequencing, with support at every step• Automated DNA extraction in as little as 30 minutes

• Streamlined, single-tube, PCR-free library construction with high conversion rates

• Accurate, sensitive quantification for reliable library pooling and clustering

• Integrated service and support throughout the workflow

Sample Sequencing Ready Library

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DNA EXTRACTIONMagNA Pure 24* and MagNA Pure 96*

Why do extraction methods matter?High-quality starting material leads to sequencing success. High-molecular-weight input DNA is essential for the creation of libraries with the 350 – 650 bp inserts required for sequencing whole human genomes on Illumina® HiSeq® and NovaSeq™ instruments.

Obtain high-quality, high-molecular-weight DNA for direct use in sequencing with the MagNA Pure 24 and MagNA Pure 96 Systems. These fully automated nucleic acid extraction instruments provide walkaway automation, require less user intervention, and minimize variability between extractions.

• Reliable DNA extraction from as little as 200 μL whole blood

• Scalable extraction for low-, mid-, or high-throughput levels

• Optimized protocols for sequencing workflows

1 Sample lysis, inactivation of nucleases, and release of nucleic acids

2 Addition of magnetic glass particles (MGP) and binding of nucleic acids to MGPs

3 Magnetic separation of MGP/nucleic acid complexes from the lysed sample

4 Multiple washing steps

5 Elution of purified nucleic acids from MGPs

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hgDNA extracted from 200 µL whole blood in a final elution volume of 100 µL. All three extraction methods yield similar DNA profiles. (Fragment Analyzer™, using the Genomic DNA 50 kbp Analysis Kit; Advanced Analytical).

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MagNA Pure 24

42 kb

MagNA Pure 96Manual

DNA LIBRARY PREPARATIONKAPA HyperPrep Kit, Adapters, and Pure Beads

Why is library preparation so important?Reduced bias improves coverage uniformity and sequencing economy. Researchers prefer mechanically sheared DNA and PCR-free library preparation for human WGS, as enzymatic tagmentation imparts greater levels of bias.

Efficient conversion is critical to the preservation of sample complexity, especially in PCR-free workflows. In addition, workflows with efficient conversion require less input DNA and are effective with broad range of challenging sample types.

Flexibility is key. Library preparation methods for human WGS must accommodate input DNA of varying quality and concentration. At the same time, the workflows must be scalable for low- to high-throughput labs while consistently yielding reliable results.

Efficiently convert samples to sequencing-competent libraries for Illumina® sequencers with the versatile, streamlined KAPA HyperPrep Library Preparation Kits (see below).

• PCR-free workflows to minimize bias

• Effective with input DNA of varied quality and amounts

• Compatible with mechanically sheared DNA

• Tunable size selection for desired insert size

Additional accessories:

• KAPA Dual-Indexed Adapters (96 adapters at 15 µM)

• KAPA Pure Beads for DNA purification and size selection

Comparison of PCR-free workflows. The KAPA HyperPrep Kit provides higher conversion rates (above) with a shorter core library preparation time (left). DNA input was 500 ng for the KAPA HyperPrep workflow and 1 µg for the TruSeq workflow. Error bars represent +/- SD.

KAPA HyperPrepPCR-Free

Adapter Ligation

Double-sided Size Selection

Post-ligation Cleanup

End repairand A-tailing

Core library prep time: ~3.0 hr

Input: 500 ng

Covaris shearing

Library

Bead Cleanup

Illumina® TruSeq® PCR-Free

Adapter Ligation

A-tailing

End repair and size selection

Post-ligation Cleanup

Post-ligation Cleanup

Core library prep time: ~4.0 hr

Input: 1 µg

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LIBRARY QUANTIFICATIONKAPA Library Quant and Roche LightCycler®**

Why is qPCR-based library quantification preferred for library QC?Sequencing capacity is maximized when sequencing-competent molecules are accurately measured with qPCR, enabling libraries to be pooled at the desired ratios.

Clustering can be optimized by quantification of library pools, further improving sequencing results.

Library quantification via qPCR leads to accurate sample pooling and optimal clustering.

A: qPCR-based library quantification methods detect only sequencing-competent molecules. In contrast, other assays detect fragments that are not sequenceable, leading to underclustering.

B: qPCR-based quantification detects all sequenceable molecules, including single-stranded configurations often created in PCR-based workflows. Other methods do not detect these molecules, leading to overclustering.

C: Quantification of libraries and library pools with the KAPA Library Quantification Kit prior to library dilution leads to uniform cluster amplification.

A: PCR-free workflow

B: Workflow with amplification

Sequencing-competentmolecules

Double-strandedlibrary molecules

Sequenceable moleculesnot detected by

fluorometric assays

Partial library fragments

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KAPA Library Quantification Kits contain all reagents needed for qPCR-based quantification of NGS libraries for Illumina® sequencing.

• Accurate quantification of sequencing-competent libraries

• Better accuracy when pooling libraries

• Automation-friendly workflow for increased throughput

Roche LightCycler® 96 and LightCycler® 480 Instruments ensure reproducible, reliable, accurate data.

• Scalable instrument options

• Dependable temperature accuracy and homogeneity

• Ideal for use with KAPA Library Quantification Kits

C:

RESOURCESSeq to Learn

Consult our dedicated Support & Applications Scientists

• Discuss project-specific workflow considerations

• Develop and install automation scripts and workflows

• Troubleshoot challenges

• Request wet-lab training

• Consult and collaborate to integrate new applications into your lab

Contact us: support.seqls@roche.com

Seq success for your project?

ASK US ABOUT OUR SEQUENCING WORKFLOW BUNDLES.Contact your rep or visit: go.roche.com/seqbundles

App Note KAPA HyperPrep Kits offer a flexible, high-efficiency library preparation solution for PCR-free human whole-genome sequencing. Roche Sequencing Solutions, 2018.

Find this and other resources at: go.roche.com/wgs

Data on file.*All MagNA Pure 24 and MagNA Pure 96 Kits, Consumables, and Accessories are for in vitro diagnostic unless otherwise noted.**For Life Science Research Use Only. Not for use in diagnostic procedures.

KAPA, LIGHTCYCLER, and MAGNA PURE 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. MC-US-01350 SS115002 A376 8/18

Published by:

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

ORDERING INFORMATION

Code Description Kit Size

07290519001 MagNA Pure 24 System 1 instrument

06541089001 MagNA Pure 96 System 1 instrument

KK8501 KAPA HyperPrep Kit, no amplification 8 rxn

KK8503 KAPA HyperPrep Kit, no amplification 24 rxn

KK8505 KAPA HyperPrep Kit, no amplification 96 rxn

KK8722 KAPA Dual-Indexed Adapter Kit, 15 µM 96 x 20 µL

KK8000 KAPA Pure Beads 5 mL

KK8001 KAPA Pure Beads 30 mL

KK8002 KAPA Pure Beads 60 mL

05815916001 LightCycler® 96 1 instrument

05015278001 LightCycler® 480 (96-well) 1 instrument

05015243001 LightCycler® 480 (384-well) 1 instrument

KK4854 KAPA Library Quantification Kit for Illumina® Platforms: LC480 qPCR Master Mix 500 rxn

KK4824 KAPA Library Quantification Kit for Illumina Platforms: Universal Master Mix 500 rxn

ASK US ABOUT OUR SEQUENCING WORKFLOW BUNDLES.Contact your rep or visit: go.roche.com/seqbundles