Nov 14, 2016
JetSeq™ DNA Library Preparation Kit
- Bioline
- Bioline
External link: http://www.bioline.com/jetseq
Protocol Citation: Bioline 2016. JetSeq™ DNA Library Preparation Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.fwgbpbw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 25, 2016
Last Modified: December 09, 2017
Protocol Integer ID: 3752
Abstract
This protocol is for the JetSeqTM DNA Library Preparation Kit. The kit is designed to generate high-quality next generation sequencing (NGS) libraries suitable for sequencing on Illumina MiSeqTM, NextSeqTM or HiSeqTM instruments.
Guidelines
1. KIT CONTENTS
Box1:
Box 2: JetSeq DNA Library Preparation Index Set (1-16, 20µl each)
2. DESCRIPTION
The success of next-generation sequencing is dependent upon the precise and accurate processing of the input DNA. This requires high-quality library preparation of sheared DNA using a coordinated series of standard molecular biology reactions whilst maintaining high yields during the intermediate puri cation steps.
The JetSeqTM DNA Library Preparation Kit is designed to generate high-quality next generation sequencing (NGS) libraries suitable for sequencing on Illumina MiSeqTM, NextSeqTM or HiSeqTM instruments. The kit contains all of the enzymes and buffers necessary for end-repair, A-tailing, ligation and amplification in convenient master mix formulations as well as 16 barcoded adapters that can be used for single or multiplex reads.
Low input: 0.01-3 μg fragmented DNA
Increased speed: sequencing ready library in under 3 hours
Improved confidence: simpler protocol improves reproducibility
Improved quality: maximum coverage from all sample types
Maximum convenience: all-in-one kit
By combining end-repair and A-tailing in one unique step, the JetSeqTM DNA Library Preparation Kit is able to reduce total NGS library preparation time and minimize the variability caused by additional handling, as well as the risk of contamination.
Please read this manual carefully to familiarize yourself with the JetSeqTM DNA Library Preparation protocol before starting (also available on www.bioline. com/jetseq).
3. STORAGE
When stored under the recommended conditions and handled correctly, full activity of reagents is retained until the expiry date indicated on the outer box label.
The kit components should be stored at -20 °C. It is recommended that the user avoid repeated freeze-thaw cycles.
4. SAFETY INFORMATION
When working with chemicals, always wear suitable personal protective equipment, including lab coat, gloves and safety glasses.
For detailed information, please consult the material safety data sheets available on our website at www.bioline.com.
5. PRODUCT SPECIFICATIONS
The JetSeqTM DNA Library Preparation Kit is designed for Illumina® library construction work ows for a wide range of NGS applications, including: targeted sequencing (capture), whole genome sequencing, de novo sequencing, whole exome sequencing and ChIP sequencing.
Fig. 1 Workflow for JetSeqTM DNA Library Preparation Kit
6. EQUIPMENT AND REAGENTS TO BE SUPPLIED BY THE USER The following additional items are required:
PCR equipment: Thermal cycler.
Equipment for the determination of DNA concentration such as NanodropTM, QubitTM, TapestationTM, Bioanalyzer or equivalent.
Equipment for the determination of DNA size distribution such as TapestationTM, Bioanalyzer or equivalent.
Equipment for the puri cation and size selection of DNA fragments such as AMPureTM, DynabeadsTM, SPRITM beads or other equivalent column-based systems.
7. IMPORTANT NOTES
7.1. DNA preparation and quality control The most important prerequisite for any NGS library preparation is high-quality DNA. Sample handling and DNA isolation procedures are therefore critical
to the success of the experiment. Residual traces of proteins, salts or other contaminants will degrade the DNA or decrease the ef ciency of the enzymatic activities necessary for optimal library preparation.
7.1.1. Recommended genomic DNA preparation method
Depending on the sample, we recommend one of the following extraction kits:
ISOLATE II Genomic DNA Kit (BIO-52066) for the preparation of genomic DNA from fresh tissues and cells.
ISOLATE II FFPE RNA/DNA Kit (BIO-52087) for the preparation of genomic DNA from FFPE tissue samples.
ISOLATE II Plant DNA Kit (BIO-52069) for isolation of genomic DNA from plants.
For more DNA extraction kits, please refer to our ISOLATE II selection tool (www.bioline.com/isolate).
7.1.2. Recommendations for DNA fragmentation
DNA can be fragmented using one of the following methods:
Mechanical fragmentation (acoustics, sonication, nebulization).
Enzymatic fragmentation.
To ensure complete fragmentation of the DNA that is needed for library preparation, only use the recommended parameters given in the manufacturer’s instructions. Check the fragmented DNA to ensure a correct size distribution is obtained.
8. PROTOCOL
8.1. End-repair
Remove the “Step 1” reagents (green cap) and the nuclease free water (blue cap) from storage (-20 °C) and allow them to thaw on ice.
1. Prepare reaction mix on ice using the volumes shown below and mix by pipetting up and down.
Table 1. End-repair reaction mix
2. Incubate for 30 min at 20 °C then 30 min at 72 °C.
3. Transfer the reaction tube on ice (4 °C).
8.2. Adaptor ligation
Remove the “Step 2” reagents (yellow cap) from storage (-20 °C) and allow them to thaw on ice.
1. Using the end-repair reaction from section 7.1 assemble the following reagents on ice. Mix by pipetting up and down.
Table 1. Adaptor ligation reaction mix
Table 2. Recommended adaptor volumes for varying starting amounts of DNA
*Note: Lower starting amounts may need further optimization for optimal ligation.
Appendix A: Adaptor indexes
The nucleotide sequences for the 16 indexes provided are detailed in the table below.
Index Number Sequence
1 AACGTGAT
2 AAACATCG
3 AGTGGTCA
4 ACCACTGT
5 GATAGACA
6 GTGTTCTA
7 TGGAACAA
8 TGGTGGTA
9 ACATTGGC
10 CAGATCTG
11 CATCAAGT
12 AGTACAAG
13 AGATCGCA
14 GACTAGTA
15 GGTGCGAA
16 TGAAGAGA
Appendix B: Low multiplexing guidelines
Illumina platform such as MiSeq and HiSeq use a red laser to sequence A/C and a green laser to sequence G/T. To ensure accurate registration of the index read, both a red and green signal must be present at each cycle. It is also important to maintain color balance where possible.
If pooling less than eight samples in the final sequencing pool we suggest using the following index combinations
Number of samples in pool Index
--------------------------------------------------------------------------------
1 Any index
2 2 & 6
3 Option A: 4, 6 & 7
Option B: 1, 11 & 16
4 Option A: 2, 6, 10 & 14
Option B: 9, 12, 15 & 16
6 Option A: 1, 2, 4, 6, 7 & 8
Option B: 2, 8, 9, 12, 15 & 16
8 Option A: 1-8
Option B: 9-16
A TECHNICAL SUPPORT AND TROUBLESHOOTING
For technical assistance or more information on this product, please email us at tech@bioline.com
B ASSOCIATED PRODUCTS
Product Size Cat. #
ISOLATE II Genomic DNA Kit 50 prep BIO-52066
ISOLATE II FFPE RNA/DNA Kit 50 prep BIO-52087
ISOLATE II Plant DNA Kit 50 prep BIO-52069
JetSeq Library Quantification Kit TBD Please enquire
C PRODUCT WARRANTY AND DISCLAIMER
Bioline warrants that its products will conform to the standards stated in its product specification sheets in effect at the time of shipment. Bioline will replace any product that does not conform to the specifications free of charge. This warranty limits Bioline’s liability to only the replacement of the product.
D TRADEMARK AND LICENSING INFORMATION
JetSeq™ was developed jointly by OGT and Bioline. JetSeq™ (Bioline Reagents Ltd), HiSeq™, MiSeq™, NextSeq™ (Illumina Inc.); Qubit® (ThermoFisher Scientific); Dynabeads™ (Dynal Inc.); AMPure™ (Backman Coulter Inc.)
Ordering Information
Product Size Cat. #
JetSeq DNA Library Preparation Kit 16 Reactions BIO-68025
End-repair
End-repair
Remove the “Step 1” reagents (green cap) and the nuclease free water (blue cap) from storage (-20) and allow them to thaw on ice.
Prepare reaction mix on ice using the volumes shown below and mix by pipetting up and down.
Table 1. End-repair reaction mix
Incubate for 30 min at 20 °C (incubation 1/2).
00:30:00
Incubate for 30 min at 72 °C (incubation 2/2).
00:30:00
Transfer the reaction tube on ice (4 °C).
Adaptor ligation
Adaptor ligation
Remove the “Step 2” reagents (yellow cap) from storage (-20 °C) and allow them to thaw on ice.
Using the end-repair reaction from section End Repair assemble the following reagents on ice. Mix by pipetting up and down.
Table 1. Adaptor ligation reaction mix
Table 2. Recommended adaptor volumes for varying starting amounts of DNA
Note
Note: Lower starting amounts may need further optimization for optimal ligation.
Incubate for 15 min at 20 °C.
00:15:00
Clean-up and size select the adaptor-ligated library. It is important at this stage to remove unwanted adaptor-dimers.
Our suggested protocol for post ligation clean-up using AMPure XP beads:
https://www.protocols.io/view/clean-up-using-ampure-xp-beads-f3ebqje.
Note
Note: Equipment and reagents are not provided, see section 6 in the Guidelines.
Assess the quality and concentration of the cleaned up adaptor-ligated DNA.
Confirm the DNA library size distribution and the absence of adaptor-dimers on a Bioanalyzer, Tapestation or equivalent. An increase of 58 bp should be measured following the ligation of the adaptors.
Determine concentration of the purified adaptor-ligated DNA using Nanodrop, Qubit or equivalent.
Note
The purified DNA can be stored at -20 °C.
Adaptor extension (PCR 1)
Adaptor extension (PCR 1)
Remove the “Step 3” reagents (orange cap) from storage (-20 °C) and allow them to thaw on ice.
Assemble the following reaction on ice using the quantities shown below. Mix by pipetting up and down.
Table 3. PCR 1 reaction mix
Run the PCR using the following conditions.
Table 4. PCR 1 cycling conditions
Table 5. Number of cycles recommended according to the amount of puri ed adaptor-ligated DNA used
Note
It is not recommended to perform >10 cycles as this will increase the percentage of duplicates.
Check the quality of the library on a Bioanalyzer, Tapestation or similar equipment.
If target selection of DNA capture is used, skip this step.
Note
This is to ensure the absence of adaptor-dimers. If adaptor-dimers are observed it is recommended that a clean-up of the adaptor extension (PCR 1) is performed in order to remove these unwanted products.
Suggestest protocol for post adaptor extension (PCR1) clean-up using AMPure XP beads in Guidelines.
Note
This is to ensure the absence of adaptor-dimers. If adaptor-dimers are observed it is recommended that a clean-up of the adaptor extension (PCR1) is performed in order to remove these unwanted products.
Determine the PCR product concentration using a Nanodrop, Qubit or equivalent.
Note
If the samples are not to be used immediately, store at -20 °C.
Adaptor completion and indexing (PCR 2)
Adaptor completion and indexing (PCR 2)
Remove the “Step 4” reagents (purple cap) from storage (-20 °C) and allow them to thaw on ice.
Prepare the following reaction mix on ice using the quantities shown below. Mix by pipetting up and down.
Table 6. Adaptor completion and indexing (PCR 2) reaction mix
Note
If target selection or DNA capture is used, the DNA may be at a too low concentration to be measured. In this case we would suggest to use 14 μL of the enriched fraction.
Run the PCR with the following cycling conditions:
Table 7. Adaptor completion and indexing (PCR 2) cycling conditions
Note
If target selection or DNA capture is used the DNA may be at a too low concentration to be measured. In this case we would suggest to use 20 cycles.
Check the quality of the library on a Bioanalyzer, Tapestation or similar equipment. This is to ensure the absence of adaptor-dimers.
- If adaptor dimers are observed it is recommended to remove these unwanted products by size selection using a suitable clean-up and size selection equipment and reagents.
- If no adaptor dimers are detected perform only a clean-up of the product of PCR 2 using a suitable clean-up and size selection equipment and reagents
Note
Note: Equipment and reagents are not provided, see section 6 of the guidelines.
Note
When comparing the products of PCR 1 (adaptor extension) and PCR 2 (adaptor completion and indexing reaction), an increase of approximatively 70 bp of the DNA size should be observed.
Determine the PCR product concentration using Nanodrop, Qubit or equivalent. For accurate measurement we recommend the JetSeq Library Quantification Kit.
The DNA library is ready for sequencing on MiSeq, NextSeq and HiSeq platforms and can be pooled if necessary.