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

SAMPLE QC& QUANTIFICATION

KAPA hgDNA Quantification and QC Kits contain all the reagents needed for the qPCR-based quantification and quality assessment of human genomic DNA samples prior to NGS library construction.

Kits contain KAPA SYBR® FAST qPCR Master Mix, optimized for high-performance SYBR Green I-based qPCR, as well as a pre-diluted set of DNA standards and primer premixes targeting different portions of a highly conserved single-copy human locus. Absolute quantification is achieved with the primer pair defining the shortest fragment, whereas the additional primers are used to derive information about the amount of amplifiable template in the DNA sample. Quality scores (or Q-ratios) generated with the kit may be used to predict the outcome of library construction, or tailor workflows for samples of variable quality, particularly FFPE DNA.

Benefits• Quantification and quality assessment with a single assay

• Ability to correlate quality scores to sequencing metrics

• High-performance KAPA SYBR FAST qPCR Master Mix

• A pre-diluted set of DNA standards and primer premixes

• A versatile and easy-to-automate workflow

KAPA hgDNA Quantification and QC KitQuality matters

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

Obtain concentration and quality information with a single assay• Allows for absolute quantification of dilute DNA samples

• Quantification with an additional primer pair provides a Q-ratio that is indicative of sample quality

Principle of the hgDNA Quantification and QC assay. A single set of DNA standards is used to generate up to three standard curves using three different primer pairs that amplify targets of 41 bp, 129 bp or 305 bp within a conserved, single-copy human locus. The 41 bp assay is used for absolute quantification of DNA samples. For an assessment of DNA quality, standard curves are generated and samples assayed with the 129 bp and/or 305 bp primer premix(es). Since poor DNA quality has a greater impact on the amplification of longer targets, the relative quality of a DNA sample can be inferred by normalizing the concentration obtained using the 129 bp or 305 bp assay against the concentration obtained from the 41 bp assay. This normalization generates a Q-ratio (with a value between 0 and 1) that is indicative of DNA quality, or the amount of amplifiable material in a DNA sample.

Damaged hgDNA

CycleCycle

High-quality hgDNA

5 10 15 20 25 30 35 40

CycleCycle

Cycle

Cycle

X X X

Conv

ersio

n (%

)

10-0.00

10-0.25

10-0.50

10-0.75

10-1.00

10-1.25

10-1.50

10-1.75

10-2.00

5 10 15 20 25 30 35 40

Nor

mal

ized

fluo

resc

ence

305 bp Q129 bp

Q41 bp<1

Q305 bp

Q41 bp<<1

Conv

ersio

n (%

)

10-0.00

10-0.25

10-0.50

10-0.75

10-1.00

10-1.25

10-1.50

10-1.75

10-2.00

5 10 15 20 25 30 35 40

Nor

mal

ized

fluo

resc

ence

305 bp Q129 bp

Q41 bp≈1

Q305 bp

Q41 bp≈1

Conv

ersio

n (%

)

10-0.00

10-0.25

10-0.50

10-0.75

10-1.00

10-1.25

10-1.50

10-1.75

10-2.00

5 10 15 20 25 30 35 40

Nor

mal

ized

fluo

resc

ence

129 bp

Conv

ersio

n (%

)

10-0.00

10-0.25

10-0.50

10-0.75

10-1.00

10-1.25

10-1.50

10-1.75

10-2.00

Nor

mal

ized

fluo

resc

ence

41 bp

Conv

ersio

n (%

)

10-0.00

10-0.25

10-0.50

10-0.75

10-1.00

10-1.25

10-1.50

10-1.75

10-2.00

5 10 15 20 25 30 35 40

Nor

mal

ized

fluo

resc

ence

41 bp

Conv

ersio

n (%

)

10-0.00

10-0.25

10-0.50

10-0.75

10-1.00

10-1.25

10-1.50

10-1.75

10-2.00

5 10 15 20 25 30 35 40

Nor

mal

ized

fluo

resc

ence

X

129 bp

X X X

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

Input Amount

Conversion rates for libraries prepared for targeted re-sequencing on the Illumina platform. Libraries were prepared in duplicate from 80 – 100 ng FFPE DNA samples of variable quality (orange) or a commercial hgDNA preparation (grey), using the KAPA LTP Library Preparation Kit. FFPE DNA samples were assessed with the KAPA hgDNA Quantification and QC assay prior to Covaris shearing, and are arranged on the x-axis in order of increasing Q-ratio (DNA quality). Q-ratios were as indicated. The amount of fragmented DNA (average size ~180 bp) used for library construction was determined using the Qubit® HS dsDNA assay (Life Technologies), whereas adapter-ligated libraries were quantified using the qPCR-based KAPA Library Quantification Kit. Results indicated that conversion rates (% input DNA converted to adapter-ligated library) for FFPE samples were significantly lower than for high-quality DNA. This limits the diversity of FFPE libraries, and necessitates more cycles of library amplification to generate sufficient material for target capture — leading to higher duplication rates and lower target coverage for FFPE libraries.

Employ quality scores in the analysis of NGS library construction workflows• Q-ratios can provide valuable insights into the bottlenecks in NGS library construction workflows

• For FFPE samples, library and sequence quality is primarily limited by inefficient conversion of input DNA to adapter-ligated library

Conv

ersio

n (%

)

40

35

30

25

20

15

10

5

0

FFPE

_1A

FFPE

_1B

FFPE

_2A

FFPE

_2B

FFPE

_3A

FFPE

_3B

FFPE

_4A

FFPE

_4B

Cont

rol_

A

Cont

rol_

B

Con

vers

ion

(%)

Establish sample quality control minimums• Indicates whether a sample is of sufficient quality for successful library preparation

• Reduces time and effort by eliminating library construction and sequencing of samples that do not pass QC

DMHC Q-ratios for sequenced and not sufficient (QNS) samples. Implementation of the KAPA hgDNA Quantification and QC Kit after DNA extraction, but prior to library preparation, enabled the user to establish an additional quality control metric that could be used to predict sequencing performance earlier in the process. A DHMC Q Ratio of Q129/41 > 0.5 and Q305/41 > 0.2 for use in amplicon sequencing workflows was empirically determined. Data shown is courtesy of the Translational Research Laboratory at Dartmouth-Hitchcock Medical Center (DHMC).

Sequ

ence

d sa

mpl

esQ

NS’

d sa

mpl

es

< 0.5 < 0.2

0.2 - 0.8

Sequenced samples(High-quality DNA)Q129bp/Q41bp: 0.5 - 1.0Q305bp/Q41bp: 0.2 - 0.8

QNS’d samples(Low-quality DNA)

Q129bp/Q41bp: <0.5Q305bp/Q41bp: <0.2

DHMC QC ratioQ129/Q41 > 0.5Q305/Q41 > 0.2

Q129 bp / Q41 bp ratio

Q129 bp / Q41 bp ratio

Q305 bp / Q41 bp ratio

Q305 bp / Q41 bp ratio1.50

1.00

0.50

0.001.20 1.00 0.800.40 0.600.200.00

1.50

1.00

0.50

0.001.00 0.800.40 0.600.200.00

1.50

1.00

0.50

0.001.20 1.00 0.800.40 0.600.200.00

1.20

1.00

0.80

0.40

0.60

0.20

0.001.20 1.00 0.800.40 0.600.200.00

0.5 - 1.0

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 product names and trademarks are the property of their respective owners. ©2017 Roche Sequencing Solutions, Inc. All rights reserved. SEQ100006 A038 02/17

Input Amount

Ordering information

Roche Cat. No. KAPA Code Description Kit Size

07960590001 KK4960 Complete kit, with Universal qPCR Master Mix 300 x 20 μL rxn

07960603001 KK4961 Complete kit, with ABI Prism qPCR Master Mix 300 x 20 μL rxn

07960611001 KK4962 Complete kit, with Bio-Rad qPCR Master Mix 300 x 20 μL rxn

07960620001 KK4963 Complete kit, with qPCR Master Mix (optimized for Roche LightCycler® 480) 300 x 20 μL rxn

07960689001 KK4969 Complete kit, with ROX Low qPCR Master Mix 300 x 20 μL rxn

Obtain actionable data for sample preparation from FFPE DNA• Q-ratios correlate with key sequencing metrics such as duplication rates and mean target coverage

• FFPE samples with a Q score >0.4 yield libraries of acceptable quality when processed with the KAPA HTP or LTP Library Preparation Kit in standard sample preparation pipelines for target capture

Q129/41 ratios correlate with key sequencing metrics, such as duplication rate. Libraries were prepared manually from 250 ng Covaris-sheared FFPE DNA (n=14) or control DNA, extracted from cultured cells, fresh-frozen tissue or blood (n=6). Samples are arranged on the x-axis (left to right) in order of increasing Q129/41-ratio (designated by orange bars for FFPE samples and blue bars for control DNAs). Libraries were constructed with NEBNext® reagents (New England Biolabs), whereas the KAPA HiFi Kit was used for pre- and post-capture amplification (10 cycles). Target capture was performed with a custom SeqCap EZ panel (300 genes, 0.9 MB). Sequencing (2 x 100 bp) was performed on an Illumina HiSeq. The average duplication rate (grey dots) were two times higher for FFPE (75% vs. 38% for control samples). FFPE samples with a Q129/41 ratio >0.4 yielded libraries that met minimum sequence quality requirements.

Conv

ersio

n (%

)

1.20

1.00

0.80

0.60

0.40

0.20

0.00

Q-r

atio

Q129/41 (FFPE)

Q129/41 (frozen/blood/cell line)Duplication rate

R2= 0.859

02_ F

FPE

Nor

mal

16_ F

FPE

Tum

or

01_ F

FPE

Tum

or

15_ F

FPE

Tum

or

19_ F

roze

nTu

mor

07_ F

FPE

Nor

mal

06_ F

FPE

Tum

or

14_ F

FPE

Nor

mal

10_ F

FPE

Tum

or

18_ F

roze

nTu

mor

09_ F

FPE

Tum

or

08_ F

FPE

Nor

mal

04_ F

FPE

Tum

or

12_ F

roze

nTu

mor

05_ F

roze

nTu

mor

03_ F

FPE

Tum

or

17_ F

FPE

Nor

mal

11_ F

FPE

Tum

or

13_ B

lood

Nor

mal

20_ C

ell li

neTu

mor

120%

100%

80%

60%

40%

20%

0%

Conv

ersio

n (%

)

1.60

1.40

1.20

1.00

0.80

0.60

0.40

0.20

0.00

1600

1400

1200

1000

800

600

400

200

Q-r

atio

02_ F

FPE

Nor

mal

16_ F

FPE

Tum

or

01_ F

FPE

Tum

or

15_ F

FPE

Tum

or

19_ F

roze

nTu

mor

07_ F

FPE

Nor

mal

06_ F

FPE

Tum

or

14_ F

FPE

Nor

mal

10_ F

FPE

Tum

or

18_ F

roze

nTu

mor

09_ F

FPE

Tum

or

08_ F

FPE

Nor

mal

04_ F

FPE

Tum

or

12_ F

roze

nTu

mor

05_ F

roze

nTu

mor

03_ F

FPE

Tum

or

17_ F

FPE

Nor

mal

11_ F

FPE

Tum

or

13_ B

lood

Nor

mal

20_ C

ell li

neTu

mor

Q129/41 (FFPE)

Q129/41 (frozen/blood/cell line)Mean target coverage

R2= 0.801

Dup

licat

ion

rate

Mea

n ta

rget

cov

erag

e (X

)