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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
)