www.toyobo.co.jp/e/bio

F1753K

Instruction manual for GenNext NGS Library Prep Kit 21033

GenNextTM NGS Library Prep Kit
LPK-101T 24 reactions
LPK-101T 8 reactions
LPK-101L 96 reactions
Store at -20°C

Contents[1]

Introduction[2]

Components[3]

Protocol1. End repair and A-tailing
2. Adapter ligation
3. Cleanup (after adapter ligation)
4. Library amplification
5. Cleanup (after library amplification)
6. Size selection (optional)[4]

Library QC[5]Application data

[6]

Troubleshooting[7]

Related products

CAUTIONAll reagents in this kit are intended for research purposes. Do not use for diagnostic or clinical purposes. Please observe
general laboratory safety precautions while using this kit.
- illumina® and MiSeq® are registered trademarks of illumina, Inc.
- Agencourt® and AMPure® are registered trademarks of Beckman Coulter, Inc.

JAPANTOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

1

www.toyobo.co.jp/e/bio[ 1 ] Introduction

Description
GenNext™ NGS Library Prep Kit comprises the enzymes and buffers for preparing
libraries for illumina® sequencing from fragmented double-stranded DNA and PCR
products.
With this system, it is possible to conveniently and quickly convert a broad range (1ng 1μg) of input amounts of DNA into libraries for illumina® sequencing. Terminal repair
and 3' end adenylation of the fragmented DNA can be conducted in the end repair andA-tailing step. Platform-specific adapters are then ligated to both ends of the DNA
fragments.
If required, a high-fidelity amplification step can be performed using the reagentsincluded in the GenNext™ NGS Library Prep Kit. Library Amplification Master Mix
uses a highly-accurate PCR enzyme developed using genetically-modified KOD DNA
polymerase. This minimizes the influence of GC bias on amplification and can amplify
various regions evenly.

Process Workflow* Adapters and beads required for cleanup after adapter ligation and library amplification are notincluded.

Features-Simple and quick operation flow
The steps from terminal repair and 3' end adenylation to adapter ligation can be
conducted in the same container. Terminal repair and adenylation at the 3' end can be
performed in 15 minutes. Adapter ligation can be done in 15 minutes. Library
amplification can be performed in cycles of 10 seconds’ annealing and 15 seconds’
extension.
-A wide range of input amount
GenNext™ NGS Library Prep Kit is compatible with various inputs from 1 ng to 1 μg.

JAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

1

www.toyobo.co.jp/e/bio-Low bias library amplification
Library Amplification Master Mix uses a highly-accurate PCR enzyme developed using
genetically-modified KOD DNA polymerase. It minimizes the influence on
GC-bias-induced amplification, and it is possible to amplify various regions evenly.

[ 2 ] Components

The kits include the following reagents, which can be used for 24 (LPK-101), 8(LPK-101T) and 96 (LPK-101L) reactions. All reagents should be stored at -20°C.
GenNextTM NGS Library Prep Kit (Code No. LPK-101, LPK-101T, LPK-101L)

End repair and A-tailing buffer*
End repair and A-tailing enzyme*
Ligation solution
Library amplification master mix
Library amplification primer mix

LPK-101
240 µL60 µL
1,200 μL
690 µL138 µLLPK-101T
80 µL
20 µL
400 µL230 µL
46 µL

LPK-101L
960 µL240 µL1,600 μL × 3
1,380 μL × 2
552 µL* Do not store the mixed solution.
End repair and A-tailing buffer
End repair and A-tailing Buffer is optimized to convert fragmented DNA to repaired
DNA having 5´-phosphorylated, 3´-dA-tailed ends. End repair and A-tailing buffer should
be used with end repair and A-tailing enzyme.
End repair and A-tailing enzyme
End repair and A-tailing enzyme is optimized to convert fragmented DNA to repaired
DNA having 5´-phosphorylated, 3´-dA-tailed ends. End repair and A-tailing enzyme
should be used with end repair and A-tailing buffer. Please pipette slowly as this isviscous.
Ligation solution
This solution contains DNA Ligase and optimized reaction buffer. Please pipette slowly
as this is viscous.
Library amplification master mix
This is a 2× concentrated master mix containing genetically-modified KOD DNA
polymerase, dNTPs (dATP, dGTP, dCTP, dTTP) and Mg2+. Various regions of the
genome can be amplified homogeneously even if these regions contain GC bias. The
resulting amplicons are suitable for next-generation sequencing analyses.
Library amplification primer mix
The primer mix (10×) is optimized to amplify illumina® libraries flanked by the P5 andP7 flow cell sequences.

JAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

2

www.toyobo.co.jp/e/bio

Required materials not included
・Thermocycler
・Fragmentation equipment or reagent
GenNext™ NGS Library Prep Kit is compatible with mechanical and enzymatic
fragmentation methods.
・Adapters for illumina®
GenNext™ NGS Library Prep Kit is compatible with adapters in which both thecluster generation sequences and sequencing are added during the ligation step, suchas those routinely used in TruSeq (illumina®) and other similar library preparation
workflows.
・SPRI (Solid Phase Reversible Immobilization) paramagnetic bead
Agencourt® AMPure® XP Beads (Beckman Coulter, cat. no. A63880, A63881) arerecommended for use with the GenNext™ NGS Library Prep Kit.
・10 mM Tris-HCl, pH 8.0 - 8.5
Dilution of adapter stock in water and elution of DNA in water is not recommended.
・Magnetic rack/stand for magnetic bead separation
・80% ethanol (freshly prepared)[ 3 ] Protocol

1.

End repair and A-tailing(1) Prepare the reaction mix in a tube or PCR plate as follows:
Component
Fragmented dsDNA
End repair and A-tailing buffer*
End repair and A-tailing enzyme mix*

Reaction volume(60 µL)
50 µL
8 µL
2 µL

* Premixes are stable for at least 24 hours at 4°C. Use the mixed solution within 24 hours.

(2) Vortex gently or mix well by pipetting. Spin down briefly and proceed immediately
to the next step.
(3) Incubate the mixture as follows:
30°C, 10 minutes
65°C, 5 minutes
4°C, hold(4) Proceed immediately with adapter ligation as described in the next protocol.

JAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

3

www.toyobo.co.jp/e/bio

2.

Adapter Ligation(1) Dilute adapter stocks in 10 mM Tris-HCl, pH 8.0 - 8.5, as follows:
FragmentedDNA
per 50 µL
End repair
and A-tailingreaction
1 µg
500 ng
250 ng
100 ng
50 ng
25 ng
10 ng
5 ng
2.5 ng
1 ng

Adapter stock
concentration

Adapter :insertmolar ratio

15 µM
15 µM
15 µM
15 µM
15 µM
7.5 µM3 µM
1.5 µM
750 nM
300 nM

10:1
20:1
40:1
100:1
200:1
200:1
200:1
200:1
200:1
200:1

(2) In the same plate/tubes in which end repair and A-tailing was performed, prepare thereaction mix as follows:
Component
End repair and A-tailing reaction product
Adapter stock
Ligation solution

Reaction volume (110 µL)
60 µL
5 µL
45 µL

(3) Mix well and centrifuge briefly.
(4) Incubate the mixture as follows:
20°C, 15 minutes
4°C, hold(5) Immediately proceed to the next process (cleanup).

3.

Cleanup (after adapter ligation)(1) Perform a 0.8× SPRI-bead-based cleanup as follows:
Component
Adapter ligation reaction productAgencourt® AMPure® XP** Ensure that beads are fully resuspended.

Volume (198 µL)
110 µL88 µL(2) Mix well by vortexing and/or pipetting up and down multiple times.

JAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

4

www.toyobo.co.jp/e/bio(3) Incubate the plate/tubes at room temperature for 5 - 15 minutes.
(4) Place the plate/tubes on a magnetic stand to capture the beads. Incubate until thesolution is clear.
(5) Carefully remove and discard the supernatant.
(6) Add 200 μl 80% ethanol in the plate/tubes on the magnetic stand.
(7) Incubate the plate/tubes on the magnetic stand at room temperature for 30 seconds.
(8) Carefully remove and discard the ethanol.
(9) Add 200 μl 80% ethanol in the plate/tubes on the magnetic stand.
(10) Incubate the plate/tubes on the magnetic stand at room temperature for 30 seconds.
(11) Carefully remove and discard as much ethanol as possible. Be careful not to disturb
the beads.
(12) Air dry the beads at room temperature for 3 - 5 minutes while the plate/tubes are on
the magnetic stand with lids open.
Notes
Overdrying the beads may reduce the yield.
(13) Remove the plate/tubes from the magnetic stand.
(14) Resuspend the beads in 25 μL elution buffer (10 mM Tris-HCl, pH 8.0 - 8.5) andincubate the plate/tubes at room temperature for 2 minutes. If proceeding with size
selection (optional: see [3] 6), resuspend the beads in 55 μL elution buffer.
(15) Place the plate/tubes the magnetic stand to capture the beads. Incubate until thesolution is clear.
(16) Transfer the supernatant to a new plate/tubes. Purified libraries can be stored at–20°C.

4.

Library Amplification(1) Prepare a reaction mix as follows:
Component
Library amplification master nix (2×)
Library amplification primer mix (10×)
Adapter-ligated libraryJAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

Reaction volume (50 μL)
25 μL
5 μL
20 μL

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

5

www.toyobo.co.jp/e/bio(2) Mix well and centrifuge briefly.
(3) Amplify using the following cycling conditions:(amplified libraries can be stored at –20°C.)
Step
Initial denaturation
Denaturation
Annealing
Extension

Temp
94°C
98°C
60°C
68°C

Duration
2 minutes
10 seconds
10 seconds
15 seconds

HOLD

4°C

-

Cycles1
Minimum
number requiredfor optimal
amplification(see below)
1

Recommended cycle numbers
Input DNA
(into end repair andA-tailing reaction)
1 μg
500 ng
250 ng
100 ng
50 ng
25 ng
10 ng
5 ng
2.5 ng
1 ng

Cycles*

0
0
0
0-2
3-5
5-6
7-9
9-11
11-13
13-15

* The optimal number of amplification cycles may be 1 - 3 cycles higher or lower,
depending on the sample type and size distribution of the input DNA.

5.

Cleanup (after library amplification)(1) Perform a 1× SPRI-bead-based cleanup as follows:
Component
Library amplification reaction productAgencourt® AMPure® XP** Ensure that beads are fully resuspended.

Volume (100 μL)
50 μL
50 μL

(2) Mix well by vortexing and/or pipetting up and down multiple times.
(3) Incubate the plate/tubes at room temperature for 5 - 15 minutes.
(4) Place the plate/tubes on a magnetic stand to capture the beads. Incubate until thesolution is clear.
(5) Carefully remove and discard the supernatant.

JAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

6

www.toyobo.co.jp/e/bio(6) Add 200 μl 80% ethanol in the plate/tubes on the magnetic stand.
(7) Incubate the plate/tubes on the magnetic stand at room temperature for 30 seconds.
(8) Carefully remove and discard the ethanol.
(9) Add 200 μl 80% ethanol in the plate/tubes on the magnetic stand.
(10) Incubate the plate/tubes on the magnetic stand at room temperature for 30 seconds.
(11) Carefully remove and discard as much ethanol as possible. Be careful not to disturb
the beads.
(12) Air dry the beads at room temperature for 3 - 5 minutes.
Notes
Overdrying the beads may reduce the yield.
(13) Remove the plate/tubes from the magnetic stand.
(14) Resuspend the beads in an appropriate volume of elution buffer (10 mM Tris-HCl,
pH 8.0 - 8.5). Incubate the plate/tubes at room temperature for 2 minutes. If
proceeding with size selection (optional: see [3] 6), resuspend the beads in 55 μL
elution buffer.
(15) Place the plate/tubes on a magnetic stand. Incubate until the solution is clear.
(16) Transfer the supernatant to a new plate/tubes. Purified, amplified libraries can be
stored at –20°C. Proceed with size selection, library quality control or sequencing,as appropriate.

6.

Size selection (optional)

If required, size selection may be performed at several points in the process workflow, forexample, prior to end repair and A-tailing, after the post ligation cleanup or after library
amplification. Size selection results in a narrower library size distribution, but at the costof a much amount of library. Below is an example of selecting a library of 250 - 450 bp.
(1) Add 0.6× SPRI beads to the library as follows:
Component
Library
Agencourt® AMPure® XP** Ensure that beads are fully resuspended.

JAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

Volume (80 μL)
50 μL
30 μL

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

7

www.toyobo.co.jp/e/bio(2) Mix well by vortexing and/or pipetting up and down multiple times.
(3) Incubate the plate/tubes at room temperature for 5 - 15 minutes.
(4) Place the plate/tubes on a magnetic stand. Incubate until the solution is clear.
(5) Transfer 75 μl the supernatant to a new plate/tubes.
(6) Add 0.13× SPRI beads to the supernatant as follows:
Component
Library (supernatant)
Agencourt® AMPure® XP** Ensure that beads are fully resuspended.

Volume (85 μL)
75 μL
10 μL

(7) Mix well by vortexing and/or pipetting up and down multiple times.
(8) Incubate the plate/tubes at room temperature for 5 - 15 minutes.
(9) Place the plate/tubes on a magnetic stand. Incubate until the solution is clear.
(10) Carefully remove and discard the supernatant.
(11) Add 200 μL 80% ethanol in the plate/tubes on the magnetic stand.
(12) Incubate the plate/tubes on the magnetic stand at room temperature for 30 seconds.
(13) Carefully remove and discard the ethanol.
(14) Add 200 μL 80% ethanol in the plate/tubes on the magnetic stand.
(15) Carefully remove and discard as much ethanol as possible. Be careful not to disturb
the beads.
(16) Air dry the beads at room temperature for 3 - 5 minutes.
Notes
Overdrying the beads may reduce the yield.
(17) Remove the plate/tubes from the magnetic stand.
(18) Add the required amount (e.g., 20 μL) of 10 mM Tris-HCl (pH 8.0 - 8.5) to theplate/tubes and incubate these at room temperature for 2 minutes.
(19) Place the plate/tubes on a magnetic stand. Incubate until the solution is clear.
(20) Transfer the supernatant to a new plate/tubes. Purified libraries can be stored at–20°C.

JAPANTOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

8

www.toyobo.co.jp/e/bio[ 4 ] Library QC

Library quantification
Library quantification can be performed using GenNext™ NGS Library Quantification
Kit (Code No. NLQ-101) or equivalent. The kit allows the specific and accuratequantification of libraries bearing P5 and P7 adapters and can be applied to flow cell
amplification.
Library quality control
Assess the quality of the library using a capillary electrophoresis device such as Agilent
BioAnalyzer or equivalent.

JAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

9

www.toyobo.co.jp/e/bio[ 5 ] Application data

1. Example of library quantification and quality control
Libraries were prepared from fragmented human genomic DNA, E. coli genomic DNA,or 100 bp DNA ladder with the GenNextTM NGS library prep kit or another company’s
Kit (Company A).
Libraries were amplified using 0-11 cycles of PCR and the size distribution checkedusing a MultiNA (Shimadzu Corporation). Library quantifications were performed using
a GenNext™ NGS library quantification kit (code NLQ-101).
There was no difference in the distribution of libraries between the GenNext™ NGS
library prep kit and the other company’s kit (Company A). For most illumina®
sequencing platforms, 2 - 4 nM for each library is the preferred starting concentration for
denaturation and dilution guidelines. These data illustrate that the GenNext™ NGS
library prep kit achieved sufficient adapter-ligated library yields, even with low input
amounts of DNA.

Adapters of approximately 60 bp are ligated to both ends of the insert DNA fragments, so the total
size of DNA fragments will increase by approximately 120 bp.

JAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

10

www.toyobo.co.jp/e/bio

2. Example of next generation sequencing results
Libraries were prepared from 1μg or 1ng of E. coli genomic DNA with the GenNextTM
NGS library prep kit or another company’s library construction kit (Company A).
Libraries prepared from 1ng DNA were amplified using 12 PCR cycles. Sequencing was
performed on an illumina® MiSeq® and data analyzed using CLC Genomics Workbench(QIAGEN / CLC bio). Sequencing reads were down-sampled to 1 million per libraryprior to analysis.
These data illustrate that GenNextTM NGS library prep kit enables high quality sequence
data.

JAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

11

www.toyobo.co.jp/e/bio[ 6 ] Troubleshooting
SymptomPresence of adapter
dimersCause
Low-quality adapter

Non-optimal adapter
concentration

Low yield

Incorrect ratio of added
magnetic beads reagent andlibrary solution
Overdrying the magnetic
beads

Solution- Avoid excessive freezing and thawing of adapter stock
solutions.
- Dilute and store adapters in 10 mM Tris-HCl (pH 8.0 8.5).
- The optimal concentration of adapter should bedetermined by preliminary tests: try the recommended
adapter concentration (see [3] 2).
- If the yield of the library is sufficient, repeat the
purification process or perform size selection (see [3] 6).
- Ratio of SPRI magnetic bead reagent and librarysolution used for purification greatly influences size
distribution and yield. Please check whether the liquidvolume proportion is accurate.
- Overdrying the magnetic beads may reduce the yield.
Air dry the beads at room temperature within 5 minutes.

[ 7 ] Related products
Product name

Package

Code No.

500 reactions

NLQ-101

NGS Library quantification for illumina®’s instrument

GenNext™ NGS library quantification kit

JAPAN
TOYOBO CO., LTD.
Tel (+81)-6-6348-3888
www.toyobo.co.jp/e/bio
tech_osaka@toyobo.jp

CHINA
TOYOBO (SHANGHAI) BIOTECH, CO., LTD.
Tel (+86)-21-58794900

FOR RESEARCH USE ONLY. NOT FOR HUMAN OR DIAGNOSTIC USE.

12