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

SINGLE-TUBE LIBRARYCONSTRUCTION

KAPA HyperPlus KitNGS library preparation. Evolved.

Benefits• DNA fragmentation and library prep in 2.5 hours

• Very flexible with respect to DNA input (1 ng – 1 μg)

• Now includes an additional End Repair and A-Tailing module for more sensitive applications with no change to the workflow

• Reduced bias and more uniform sequence coverage

• PCR-free workflows from lower inputs

• Industry-leading conversion rates (library prep efficiency) and library complexity, particularly for FFPE DNA

• Flexibility with respect to fragment size, adapter design and library amplification

• Qualified automation methods

• Complete library prep solution with KAPA Adapters and KAPA HyperPure Beads (sold separately)

The KAPA HyperPlus Kit provides a streamlined workflow, in which DNA fragmentation and library construction are performed in a single tube. This integrated solution combines the industry-leading library construction efficiency and library quality of the KAPA HyperPrep Kit with the speed and convenience of enzymatic fragmentation.

Sample Sequencing Ready Library

Automation & Connectivity

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

Integrated fragmentation and library preparation solutionThe KAPA HyperPlus Kit includes low-bias enzymatic fragmentation, eliminating the need for mechanical DNA shearing methods that require expensive instrumentation and are difficult to automate.

• Fragment DNA and construct libraries in 2.5 hours with a single-tube, automation-friendly workflow

• High success rates from a wide range of DNA input amounts and sample types, including challenging samples such as FFPE

• Suitable for a variety of sequencing applications, including human exome and microbial whole genome sequencing

Tunable and reproducible fragmentation• Adjust library insert sizes from 150 – 800 bp by varying fragmentation time

• Reproducible insert sizes across a range of GC content and DNA input amounts

200

150

0

35 100 150 200 300 400 500 600 1000 2000 10380

100

50

10 ng 30 min100 ng 15 min10 ng 15 min

100 ng 30 min

Library fragment size distribution

bp

Fluo

resc

ence

Reproducible library fragment size distributions are obtained with different DNA inputs. Various input amounts of Escherichia coli gDNA were processed using the KAPA HyperPlus Kit with fragmentation times of 15 or 30 minutes at 37ºC. After library amplification and a single bead cleanup, samples were analyzed using an Agilent® High Sensitivity DNA Assay.

C.difficile 10 ng

E.coli 10 ng

Human 10 ng

B.pertussis 50 ngB.pertussis 10 ng

C.difficile 50 ngP.falciparum 50 ng

E.coli 50 ng

Human 50 ng

5 min 15 min 45 min

700

800

600

500

400

300

200

100

0

% G

CReproducible fragment sizes

Fragmentation time

Ave

rage

siz

e (b

p)

Defined fragmentation parameters yield consistent library insert sizes for samples from multiple species across a wide range of GC content. 10 ng or 50 ng of Bordetella pertussis (68% GC), Clostridium difficile (29% GC), Escherichia coli (51% GC), Plasmodium falciparum (20% GC) or human gDNA were fragmented for 5, 15 or 45 minutes. These fragmentation times yielded average library insert sizes of approximately 700 bp, 350 bp and 200 bp, respectively; irrespective of GC content and input amount. All fragmentation reactions were performed at 37ºC.

Total time: ~2.5 hours

Fragmentation

End Repair and A-tailing

Bead Cleanup

Adapter Ligation

Total time: ~2.5 hours NEBNext Ultra II FS

Fragmentation and A-tailing

Fragmentation and A-tailing

Sing

le tu

be

Adapter Ligation

User Digestion

Sing

le tu

be

Sing

le tu

beSi

ngle

tube

KAPA HyperPlus

Bead Cleanup

Sing

le tu

be

Library Amplification

Bead Cleanup

Sing

le tu

be

Library Amplification

Bead Cleanup

Total time: ~3.0 hours QIASeq FX

Adapter Ligation

Library Amplification

Bead Cleanup

Bead Cleanup

Bead Cleanup

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

0

1

2

3

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0

5

10

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40

1 ng 10 ng 100 ng

Post-ligation yield

KAPA HyperPlus (Existing)*KAPA HyperPlus (Upgraded)* Covaris + KAPA HyperPrep

NEBNext Ultra II FS

nM

Industry-leading library yieldsHigh conversion rates, defined as % input DNA converted to sequenceable, adapter-ligated library, lead to higher PCR-free yields, which ultimately determines library diversity and quality.

• Low-input samples no longer require specialist library construction workflows or reagents

• PCR-free workflows possible from as little as 50 ng starting material

• Both the existing and upgraded KAPA HyperPlus chemistry produce the highest post-ligation yields

Enable superior sequencing results• Higher post-ligation yields result in fewer amplification cycles and lower duplication rates

• Detect low-frequency mutations with high confidence due to decreased sequencing artifacts and increased sensitivity with the upgraded version of the KAPA HyperPlus Kit

KAPA HyperPlus Kits produce the highest yields from a range of input amounts. The KAPA HyperPlus Kit (both existing and upgraded workflows) converts more input DNA to adapter-ligated library than NEBNext® Ultra II FS Kits or Covaris shearing combined with KAPA HyperPrep. PCR-free libraries were prepared from 1 ng, 10 ng and 100 ng E.coli gDNA using KAPA UDI Adapters and KAPA HyperPure Beads for all workflows, including NEBNext Ultra II FS Kits. Libraries were quantified after ligation using the KAPA Library Quantification Kit.

Input Amount

* KAPA HyperPlus (existing) represents the use of the HyperPrep End Repair & A-Tailing Enzyme Mix (original enzyme); KAPA HyperPlus (upgraded) represents the use of the HyperPlus End Repair & A-Tailing Enzyme Mix (a new enzyme that is now included in the KAPA HyperPlus Kit).

Lower duplication rates. Whole human exome libraries were prepared using 100 ng inputs with the exisiting and upgraded KAPA HyperPlus Kit, or the KAPA HyperPrep Kit. Captures were performed with the SeqCap EZ MedExome panel.

Improved sequencing performance. Whole human exome libraries were prepared using 100 ng inputs with the existing and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel. Strand-split artifact reads (SSARs) represent chimeric reads that appear to be derived from non-contiguous portions of the genome.1

Higher single nucleotide polymorphism (SNP) sensitivity. Whole human exome libraries were prepared using 100 ng high-quality gDNA with the KAPA HyperPrep Kit, the existing and upgraded KAPA HyperPlus Kit. Captures were performed with the SeqCap EZ MedExome panel.

10

12

14

16

18

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22

24

26

gDNA FFPE

Duplication rates

(%)

KAPA HyperPlus (Existing)*KAPA HyperPlus (Upgraded)*Covaris + KAPA HyperPrep

0

0.1

0.2

0.3

0.4

0.5

gDNA FFPE

Sequencing qualityStrand-Split Artifact Reads (SSAR)

(%)

KAPA HyperPlus (Existing)*KAPA HyperPlus (Upgraded)*

95

96

97

98

99

100

Sensitivity

(%)

KAPA HyperPlus (Existing)*KAPA HyperPlus (Upgraded)*Covaris + KAPA HyperPrep

Published by:

Roche Sequencing Solutions, Inc.4300 Hacienda DrivePleasanton, CA 94588

sequencing.roche.com

Data on file.For Research Use Only. Not for use in diagnostic procedures.KAPA and SeqCap are trademarks of Roche. All other trademarks are the property of their respective owners. ©2019 Roche Sequencing Solutions, Inc. All rights reserved. MC--01738 08/19

Ordering information

Minimal sequence coverage bias• Lower sequence bias when compared other enzymatic fragmentation methods

• Less bias leads to more uniform sequencing coverage and reduced sequencing costs

Roche Cat. No. KAPA Code Description Kit Size

07962380001 KK8510 KAPA HyperPlus Kit with Library Amplification 8 rxn

07962401001 KK8512 KAPA HyperPlus Kit with Library Amplification 24 rxn

07962428001 KK8514 KAPA HyperPlus Kit with Library Amplification 96 rxn

07962398001 KK8511 KAPA HyperPlus Kit, PCR-free 8 rxn

07962410001 KK8513 KAPA HyperPlus Kit, PCR-free 24 rxn

07962436001 KK8515 KAPA HyperPlus Kit, PCR-free 96 rxn

GC bias comparison. GC bias for C.difficile (left), E.coli (middle) and B.pertussis (right) was assessed by calculating the GC content of the reference in 100 bp bins and plotting normalized coverage across these bins for the KAPA HyperPlus, NEBNext® Ultra™ FS and QIAseq FX workflows, using Picard CollectGCBiasMetrics. Libraries were prepared from 10 ng of input DNA. In the absence of sequencing bias, all bins would be equally represented, indicated by a horizontal distribution centered on a normalized coverage of 1. Distribution of GC content in the genome is indicated by the grey histograms.

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50

2.01.81.61.41.21.00.80.60.40.2

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% GC for 100 bp windows

Nor

mal

ized

cov

erge

Num

ber of window

s (x103)

C.difficile (29% GC)

KAPA HyperPlus NEBNext Ultra II FS QiaSeq FX

30

2.01.81.61.41.21.00.80.60.40.2

032 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 70

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% GC for 100 bp windows

Nor

mal

ized

cov

erge

Num

ber of window

s (x103)

KAPA HyperPlus NEBNext Ultra II FS QiaSeq FX

E.coli (51% GC)

45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85

2.01.81.61.41.21.00.80.60.40.2

0 0

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% GC for 100 bp windows

Nor

mal

ized

cov

erge

Num

ber of window

s (x103)

KAPA HyperPlus NEBNext Ultra II FS QiaSeq FX

B.pertussis (68% GC)

1 Source: Haile, et al. (2019) Sources of erroneous sequences and artifact chimeric reads in next generation sequencing of genomic DNA from formalin-fixed paraffin-embedded samples. Nucleic Acids Research, 2019, 47,2. doi: 10.1093/nar/gky1142.