NEB 0.01 0.1 1 10 0.0002 0.002 0.02 0.2 2 20 10 12 14 16 18 20 22 24 26 28 C q KAPA Standard Concentration (pM) Nathan A. Tanner, Janine G. Borgaro, Erbay Yigit, Don Johnson, Julie F. Menin, Eileen T. Dimalanta and Nicole M. Nichols New England Biolabs, Ipswich, MA 01938, USA Benefits of Illumina® Library Quant a on with NEBNe xt® Library Quant Kit Introd ucon Accurate quantitation of a next-generation sequencing library is essential to maximizing data output and quality from each instrument run. qPCR is widely accepted as the most effective method for library quantitation by both users and manufacturers 1 , as qPCR methods measure only sequenceable library fragments with a high level of accuracy and consistency. The NEBNext Library Quant Kit for Illumina presents a simple, robust method for quantitation of Illumina libraries. Here we demonstrate the effectiveness of the Kit for a broad range of library types and sizes as well as advantages offered by qPCR quantitation for obtaining optimal cluster density and user-to-user consistency. The NEBNext Quant Kit offers an efficient and cost-effective qPCR library quantitation workflow for users looking to optimize both sequencing yield and throughput. 1: Introduction 3: Library Quant aon Consist ency 4: Accurat e Library Quant aon Produces Opmal Clust er Density www.NEBNext.com NEBNext Library Quant Kit Performance Across Library Type and Size 5: Cluster Density from Various Quant ated Libraries 6: Range of Library Sizes and Types Quant ated www.neb.com www.NEBNext.com Sequencing Cluster Density using Quant Kits Seven different Illumina libraries were quantitated using either the NEBNext (orange) or KAPA (grey) library quantitation kit. Undiluted library concentration ranged from 2–200 nM, and libraries were diluted to 8 pM and loaded onto a MiSeq™ instrument (v2 chemistry; MCS v2.4.1.3). Libraries quantitiated with the NEBNext kit resulted in a raw cluster density average of 1160 k/mm 2 , directly in the optimal range of 900–1300 k/mm 2 . In contrast, libraries loaded based on the KAPA quantitation averaged only 660 k/mm 2 . www.NEBNext.com NEBNext ® is a registered trademarks of New England Biolabs, Inc. Illumina ® , TruSeq™ and MiSeq™ are trademarks of Illumina, Inc. Bioanalyzer™ is a trademark of Agilent Technologies, Inc. KAPA™ is a trademark of Kapa Biosystems. Introd ucon 2: Quant Kit Workflow and qPCR Data The NEBNext Quant Kit uses 4 pre-diluted DNA standards and Illumina adaptor-specific primers to quantitate diluted library samples of interest. The use of 4 standards maximizes the number of libraries that can be quantitated without sacrificing performance. Example data from the same library quantitated with two different qPCR instruments are shown, with a consistent determination of library concentration. Quant Reproducibility Three 340–400 bp libraries were quantitated by 4 different users 2–4 times using either the NEBNext or KAPA™ Library Quant Kit. A marked improvement in quantitation consistency was observed for concentrations determined by the NEBNext Library Quant Kit (orange) versus those from the KAPA kit (gray). qPCR v. Bioanalyzer™ Concentrations of 4 libraries were determined by NEBNext Library Quant Kit (orange) and compared to values measured by Bioanalyzer (blue). Compared to NEBNext qPCR, Bioanalyzer concentrations displayed a greater level of variation. This finding demonstrates the benefits of qPCR for library quantitation. 7: Summary Libraries from 150–963 bp from indicated genomic input were first quantitated using the NEBNext Library Quant Kit, then diluted to 8 pM and loaded onto a MiSeq (v2 chemistry; MCS v2.4.1.3). Library concentrations ranged from 2–140 nM, but resulting raw cluster density for all libraries was 790–1300 k/mm 2 (ave. =1160). Optimal cluster density was achieved using concentrations determined by the NEBNext Library Quant Kit for all library sizes and gDNA or sRNA inputs. A selection of libraries successfully quantitated with the NEBNext Library Quant Kit. Libraries are plotted by size, ranging from smaller libraries (sRNA, FFPE) at 150–230 bp to largest libraries at 980 bp. Various source gDNA were used (e.g. human from FFPE, PCR-Free, ImmuneSeq, IMR- 90, Jurkat), ranging from 20–70% GC content as indicated by text color. Libraries were prepared with NEBNext, Illumina TruSeq™, and KAPA library prep kits. No dependence on size or GC content was observed in library quantitation. • NEBNext Library Quant Kit provides reliable qPCR library quantitation of Illumina libraries • Library quants are more reproducible and consistent vs. Bioanalyzer, KAPA • Accurate library quant produces optimal sequencing cluster density • Easy-to-use quant tool at NEBiocalculator.neb.com • NEBNext Library Quant Kit accommodates libraries 150–1000 bp, 20–70% GC, various prep methods 1: Illumina (2015). Diagnosing and Preventing Flow Cell Overclustering on the MiSeq System, p.8. (http://support.illumina.com/content/dam/illumina-marketing/documents/products/other/miseq- overclustering-primer-770-2014-038.pdf) Jurkat (633 bp) E.coli P. falciparum NEB Bioanalyzer 0 20 40 60 80 100 120 140 Library Quant (nM) NEB Bioanalyzer NEB Bioanalyzer NEB Bioanalyzer Jurkat (924 bp) Standards Lot-to-lot Consistency Accurate qPCR quantitation requires the use of high- quality DNA standards with known concentration. The NEBNext Library Quant Kit contains 4 standards produced with a high level of both quantitation accuracy and consistency. Above is data from 70+ total runs of 4 lots of both NEB and KAPA standards, with all C q plotted. Box and whiskers indicate mean and quartiles. The NEBNext Library Standards displayed much lower variation in C q , resulting in more consistent quantitation performance. User 1 User 2 User 3 User 4 NEB KAPA NEB KAPA NEB KAPA NEB KAPA E.coli H. influenzae IMR-90 100 200 qPCR Quant (nM) 100 200 qPCR Quant (nM) 100 200 qPCR Quant (nM) 4 Lots N=70 4 Lots N=72 500 600 700 800 900 1000 1100 1200 1300 Raw Cluster Density (k/mm 2 ) 0 50 100 150 200 qPCR Quant (nM) qPCR Quant Method NEBNext KAPA Target Density 0 50 100 qPCR Quant (nM) 0 400 800 1200 Cluster Density (k/mm 2 ) Input: Library Size: sRNA R. palustris R. palustris R. palustris H. influenzae H. influenzae E. coli E. coli H. influenzae Jurkat Jurkat 153 bp 310 311 341 633 638 656 900 946 958 963 Target Density Library Size 0 bp 1000 200 20% 70% GC Content 400 600 800 sRNA (A. thaliana) sRNA (human) P. falciparum H. influenzae H. influenzae H. influenzae FFPE PCR-Free IMR-90 E. coli E. coli E. coli BEI Mix R. palustris R. palustris R. palustris ImmuneSeq Jurkat Jurkat Prepare Reagents Dilute Libraries Set up Reactions Run qPCR Analyze Data Time 5 min. 10 min. 20 min. 60 min. 10 min. Total: 1h 45 min Standards NTC Library Standards NTC Library Size adjusted library concentration= 69.8 nM Size adjusted library concentration= 69.6 nM Bio-Rad CFX96 Touch™: Linear amplification plot, No ROX normalization Applied Biosystems® 7500 Fast: log amplification plot, ROX
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NEB
0.01 0.1 1 10 0.0002 0.002 0.02 0.2 2 20
10
12
14
16
18
20
22
24
26
28
Cq
KAPA
Standard Concentration (pM)
Nathan A. Tanner, Janine G. Borgaro, Erbay Yigit, Don Johnson, Julie F. Menin, Eileen T. Dimalanta and Nicole M. Nichols
New England Biolabs, Ipswich, MA 01938, USA
Benefits of Illumina® Library Quantitation with NEBNext® Library Quant Kit
Introduction
Accurate quantitation of a next-generation sequencing library is essential tomaximizing data output and quality from each instrument run. qPCR is widelyaccepted as the most effective method for library quantitation by both users andmanufacturers1, as qPCR methods measure only sequenceable library fragmentswith a high level of accuracy and consistency. The NEBNext Library Quant Kit forIllumina presents a simple, robust method for quantitation of Illumina libraries.Here we demonstrate the effectiveness of the Kit for a broad range of librarytypes and sizes as well as advantages offered by qPCR quantitation for obtainingoptimal cluster density and user-to-user consistency. The NEBNext Quant Kitoffers an efficient and cost-effective qPCR library quantitation workflow for userslooking to optimize both sequencing yield and throughput.
1: Introduction
3: Library Quantitation Consistency 4: Accurate Library Quantitation Produces Optimal Cluster Density
www.NEBNext.com
NEBNext Library Quant Kit Performance Across Library Type and Size
5: Cluster Density from Various Quantitated Libraries 6: Range of Library Sizes and Types Quantitated
www.neb.comwww.NEBNext.com
Sequencing Cluster Density using Quant KitsSeven different Illumina libraries were quantitated using either the NEBNext (orange) or KAPA (grey) library quantitation kit. Undiluted library concentration ranged from 2–200 nM, and libraries were diluted to 8 pM and loaded onto a MiSeq™instrument (v2 chemistry; MCS v2.4.1.3). Libraries quantitiatedwith the NEBNext kit resulted in a raw cluster density average of 1160 k/mm2, directly in the optimal range of 900–1300k/mm2. In contrast, libraries loaded based on the KAPA quantitation averaged only 660 k/mm2.
www.NEBNext.comNEBNext® is a registered trademarks of New England Biolabs, Inc.Illumina®, TruSeq™ and MiSeq™ are trademarks of Illumina, Inc.Bioanalyzer™ is a trademark of Agilent Technologies, Inc.KAPA™ is a trademark of Kapa Biosystems.
Introduction
2: Quant Kit Workflow and qPCR Data
The NEBNext Quant Kit uses 4 pre-diluted DNA standards and Illumina adaptor-specific primers to quantitate diluted library samples of interest. The use of 4 standards maximizes the number of libraries that can be quantitated without sacrificing performance. Example data from the same library quantitated with two different qPCR instruments are shown, with a consistent determination of library concentration.
Quant ReproducibilityThree 340–400 bp libraries were quantitated by 4 different users 2–4 times using either the NEBNext or KAPA™ Library Quant Kit. A marked improvement in quantitation consistency was observed for concentrations determined by the NEBNext Library Quant Kit (orange) versus those from the KAPA kit (gray).
qPCR v. Bioanalyzer™Concentrations of 4 libraries were determined by NEBNext Library Quant Kit (orange) and compared to values measured by Bioanalyzer (blue). Compared to NEBNext qPCR, Bioanalyzerconcentrations displayed a greater level of variation. This finding demonstrates the benefits of qPCR for library quantitation.
7: Summary
Libraries from 150–963 bp from indicated genomic input were first quantitated using the NEBNext Library Quant Kit, then diluted to 8 pM and loaded onto a MiSeq (v2 chemistry; MCS v2.4.1.3). Library concentrations ranged from 2–140 nM, but resulting raw cluster density for all libraries was 790–1300 k/mm2 (ave. =1160). Optimal cluster density was achieved using concentrations determined by the NEBNext Library Quant Kit for all library sizes and gDNA or sRNA inputs.
A selection of libraries successfully quantitated with the NEBNext Library Quant Kit. Libraries are plotted by size, ranging from smaller libraries (sRNA, FFPE) at 150–230 bp to largest libraries at 980 bp. Various source gDNA were used (e.g. human from FFPE, PCR-Free, ImmuneSeq, IMR-90, Jurkat), ranging from 20–70% GC content as indicated by text color. Libraries were prepared with NEBNext, Illumina TruSeq™, and KAPA library prep kits. No dependence on size or GC content was observed in library quantitation.
• NEBNext Library Quant Kit provides reliable qPCR library quantitation of Illumina libraries
• Library quants are more reproducible and consistent vs. Bioanalyzer, KAPA
• Accurate library quant produces optimal sequencing cluster density
• Easy-to-use quant tool at NEBiocalculator.neb.com• NEBNext Library Quant Kit accommodates libraries
150–1000 bp, 20–70% GC, various prep methods
1: Illumina (2015). Diagnosing and Preventing Flow Cell Overclustering on the MiSeq System, p.8.(http://support.illumina.com/content/dam/illumina-marketing/documents/products/other/miseq-overclustering-primer-770-2014-038.pdf)
Jurkat (633 bp) E.coli P. falciparum
NEB Bioanalyzer
0
20
40
60
80
100
120
140
Libr
ary
Qua
nt (n
M)
NEB Bioanalyzer NEB Bioanalyzer NEB Bioanalyzer
Jurkat (924 bp)
Standards Lot-to-lot ConsistencyAccurate qPCR quantitation requires the use of high-quality DNA standards with known concentration. The NEBNext Library Quant Kit contains 4 standards produced with a high level of both quantitation accuracy and consistency. Above is data from 70+ total runs of 4 lots of both NEB and KAPA standards, with all Cq
plotted. Box and whiskers indicate mean and quartiles. The NEBNext Library Standards displayed much lower variation in Cq, resulting in more consistent quantitation performance.
User 1 User 2 User 3 User 4
NEB KAPA NEB KAPA NEB KAPA NEB KAPA
E.c
oli
H. i
nflu
enza
eIM
R-9
0
100
200
qPC
R Q
uant
(nM
)
100
200
qPC
R Q
uant
(nM
)
100
200
qPC
R Q
uant
(nM
)
4 LotsN=70
4 LotsN=72
500 600 700 800 900 1000 1100 1200 1300Raw Cluster Density (k/mm2)
0
50
100
150
200
qPCR
Qua
nt (n
M)
qPCR Quant MethodNEBNext
KAPA
Target Density
0
50
100
qPC
R Q
uant
(nM
)
0
400
800
1200
Clu
ster
Den
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(k/m
m2 )
Input:Library Size:
sRNA R. palustris R. palustris R. palustrisH. influenzae H. influenzaeE. coli E. coli H. influenzaeJurkat Jurkat153 bp 310 311 341 633 638 656 900 946 958 963
Target Density
Library Size
0 bp 1000200
20% 70%GC Content
400 600 800sRNA
(A. thaliana)
sRNA (human)
P. falciparumH. influenzae
H. influenzae
H. influenzae
FFPE
PCR-Free
IMR-90E. coli
E. coli
E. coli
BEI Mix
R. palustris
R. palustris
R. palustrisImmuneSeq
Jurkat Jurkat
Prepare Reagents
Dilute Libraries
Set up Reactions
Run qPCR
Analyze Data
Time5 min.
10 min.
20 min.
60 min.
10 min.
Total: 1h 45 min
StandardsNTCLibrary
StandardsNTCLibrary
Size adjusted library concentration=69.8 nM
Size adjusted library concentration=69.6 nM
Bio-Rad CFX96 Touch™: Linear amplification plot, No ROX normalization
Applied Biosystems® 7500 Fast: log amplification plot, ROX
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