NEXTflex™ Bisulfite Library Prep Kit for Illumina Sequencing

Ideal for Illumina library prep for reduced representation bisulfite sequencing (RRBS) and whole-genome bisulfite sequencing (WGBS) 

Catalog# Product Name Quantity US List Price
5119-01 NEXTflex™ Bisulfite Sequencing Kit 8 rxns $358 Buy Now
5119-02 NEXTflex™ Bisulfite Sequencing Kit 48 rxns $1,719 Buy Now
511911 NEXTflex™ Bisulfite-Seq Barcodes - 6 48 rxns $638 Buy Now
511912 NEXTflex™ Bisulfite-Seq Barcodes - 12
96 rxns $1,101 Buy Now
511913  NEXTflex™ Bisulfite-Seq Barcodes - 24 192 rxns $1,982 Buy Now
511921 NEXTflex™ Msp1 Restriction Enzyme
8 rxns $174 Buy Now
511922 NEXTflex™ Msp1 Restriction Enzyme 48 rxns $939 Buy Now

 

 NEXTflex Bisulfite-Seq Kit box

  • Compatible with total bisulfite sequencing and reduced representation
  • Single nucleotide resolution of methylation sites
  • Uracil insensitive polymerase designed for bisulfite-converted DNA
  • Methylome-level assessment with broad genome coverage
  • Enhanced adapter ligation technology with NEXTflex™ Ligation
  • Bead-based cleanup
  • Automation-friendly workflow is compatible with liquid handlers
  • Methylated barcoded adapters for multiplexing contain embedded index sequence
  • Functionally validated with Illumina sequencing platforms

 

 

 

 

The NEXTflex™ Bisulfite-Seq Library Prep Kit is designed for both reduced representation bisulfite sequencing (RRBS) and whole-genome bisulfite sequencing (WGBS). It generates single, paired-end and multiplexed DNA libraries for sequencing on Illumina® platforms. The NEXTflex™ U+ Polymerase Mix included in the kit is able to read through uracil present in DNA templates and has been optimized to amplify bisulfite converted templates.


Complete Methylome Analysis

The NEXTflex™ Bisulfite-Seq Kit is a versatile kit designed to facilitate assessment of the methylation state of the genome and simplify workflow by using master mixed reagents and magnetic bead based cleanup to reduce pipetting and eliminate time-consuming steps in library preparation. In addition, the availability of up to 24 methylated NEXTflex™ Bisulfite-Seq barcoded adapters makes multiplexing simple. This kit features Enhanced Adapter Ligation Technology, resulting in library preps with a larger number of unique sequencing reads. This specially designed NEXTflex ligation enzymatic mix allows users to perform ligations with longer adapters and superior ligation efficiencies.


Focused Sequence Depth

The NEXTflex Bisulfite-Seq Kit with reduced representation utilizes a restriction enzyme that leads to selective amplification of CpG regions, resulting in focused sequence depth. The NEXTflex™ Msp1 Restriction Enzyme, which is available separately, has been optimized for reduced representation studies using the NEXTflex Bisulfite-Seq Kit. The NEXTflex Bisulfite-Seq Kit can also be used without the MSP 1 Restriction Enzyme for genome-wide methyl-seq analysis, including under-represented CpG regions. 

 


Papers that Cite the use of the NEXTflex Bisulfite-Seq Kit

Agarwal, P., et al. (2015) CGGBP1 mitigates cytosine methylation at repetitive DNA sequences. BMC Genomics, 16:390. doi:10.1186/s12864-015-1593-2.

Berg, A., et al. (2015) Obesity is Associated With DNA Methylation in Population-Based Adolescents. Circulation. Poster: 131:AP260, Session Title: Obesity.

Bewick, A., et. al. (2016) On the origin and evolutionary consequences of gene body DNA methylation. PNAS. 113: 9111 - 9116.

Ramesh, V., et. al. (2016) Loss of Uhrf1 in neural stem cells leads to activation of retroviral elements and delayed neurodegeneration. Genes & Development. 30: 2199 - 2212.

Satgé, C. et. al. (2016) Reprogramming of DNA methylation is critical for nodule development in Medicago truncatula. Nature Plants 2, 16166. doi:10.1038/nplants.2016.166.

Ye, R., et al. (2015) A Dicer-Independent Route for Biogenesis of siRNAs that Direct DNA Methylation in Arabidopsis. Molecular Cell. doi:10.1016/j.molcel.2015.11.015.


Kit Specs

The NEXTflex Bisulfite-Seq Kits contain enough material to prepare 8 or 48 genomic DNA samples for Illumina compatible sequencing. The shelf life of all reagents is 6 months when stored properly. DNA Binding Buffer and DNA Wash Buffer should be stored at room temperature. All of the other components can be safely stored at -20°C.

NEXTflex™ Bisulfite-Seq Protocol

NEXTflex™ Bisulfite-Seq Kit Protocol

Kit Contents

NEXTflex™ Bisulfite-Seq End Repair Buffer Mix 
NEXTflex™ Bisulfite-Seq End Repair Enzyme Mix 
NEXTflex™ Bisulfite-Seq Adenylation Mix 
NEXTflex™ Bisulfite-Seq Ligation Mix
NEXTflex™ Bisulfite-Seq DNA Adapter 24 (25 µM)
NEXTflex™ Adapter Dilution Buffer
NEXTflex™ Primer Mix (12.5 µM)
NEXTflex™ Bisulfite-Seq U+ PCR Master Mix
Nuclease-free water
Resuspension Buffer

Required Materials not Provided

10 ng - 1 µg of fragmented /or/ Msp1 digested genomic DNA in up to 40 µL nuclease-free water.
NEXTflex™ Bisulfite-Seq Barcodes – 6 / 12 / 24 (Cat # 511911, 511912, 511913 ) (Optional)
NEXTflex™ Msp1 – 8 reactions / 48 reactions (Bioo Scientific, Cat # 511921, 511922)
EZ DNA Methylation-Gold™ Kit (Zymo Research, Cat # D5005)
Ethanol 100% (room temperature)
Ethanol 80% (room temperature)
96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) / or / similar 
96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) / or / similar
Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)
Agencourt AMPure XP 5 mL (Beckman Coulter Genomics, Cat # A63880)
Magnetic Stand - 96 (Ambion, Cat # AM10027) / or / similar
Heat block
Thermocycler 
2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes
Nuclease-free barrier pipette tips
Microcentrifuge 
1.5 mL nuclease-free microcentrifuge tubes
Vortex

RRBS

reduced representation bisulfite seq bioanalyzer trace 10 nanograms

10 ng of Msp1 digested DNA input, 18 PCR cycles, 10 nm yield

 

reduced representation bisulfite seq bioanalyzer trace 1 microgram

1 ug Msp1 digested DNA input, 18 PCR cycles, 32 nm yield

 

WGBS

whole genome bisulfite seq bioanalyzer trace 10 nanograms

10 ng of 200 bp sheared DNA input, 18 PCR cycles, 58 nm yield

 

WGBS bioanalyzer trace for one microgram DNA

1 ug 200 bp sheared DNA input, 15 PCR cycles, 28 nm yield

 

Do you have protocols available for whole-genome bisulfite sequencing or reduced-representation bisulfite sequencing?

Yes, the NEXTflex™ Bisulfite-Seq Kit can be used for either WGBS or RRBS.

 

How much input DNA can be used with the NEXTflex Bisulfite-Seq Kit?

10 ng -1 µg of fragmented genomic DNA (for WGBS) or Msp1 digested genomic DNA (for RRBS) in up to 40 µL nuclease-free water.

 

How many samples can be multiplexed using the NEXTflex Bisulfite-Seq Kit? Are the indexed adapters available separately?

The methylated NEXTflex™ Bisulfite-Seq Barcodes are recommended for use with the NEXTflex Bisulfite-Seq Kit.  To offer greater flexibility, these indexed adapters are sold separately from the library preparation kits in sets of 6 or 12 unique barcodes. The 8 reaction size of the NEXTflex Bisulfite-Seq Kit (cat # 5119-01) does contain a single adapter for those customers who do not need to multiplex samples. The other barcode kits we offer are not methylated and are not compatible with bisulfite sequencing.

 

Do you recommend a bisulfite conversion kit?

We recommend the EZ DNA Methylation-Gold™ Kit (Zymo Research, Cat # D5005)

 

What is the general efficiency of bisulfite conversion using the recommended bisulfite conversion kit?

Conversion efficiency generally exceeds 99% using the EZ DNA Methylation Kit.




Do you have protocols available for whole-genome bisulfite sequencing or reduced-representation bisulfite sequencing?

Yes, the NEXTflex™ Bisulfite-Seq Kit can be used for either WGBS or RRBS.

 

How much input DNA can be used with the NEXTflex Bisulfite-Seq Kit?

10 ng -1 µg of fragmented genomic DNA (for WGBS) or Msp1 digested genomic DNA (for RRBS) in up to 40 µL nuclease-free water.

 

How many samples can be multiplexed using the NEXTflex Bisulfite-Seq Kit? Are the indexed adapters available separately?

The methylated NEXTflex™ Bisulfite-Seq Barcodes are recommended for use with the NEXTflex Bisulfite-Seq Kit.  To offer greater flexibility, these indexed adapters are sold separately from the library preparation kits in sets of 6 or 12 unique barcodes. The 8 reaction size of the NEXTflex Bisulfite-Seq Kit (cat # 5119-01) does contain a single adapter for those customers who do not need to multiplex samples. The other barcode kits we offer are not methylated and are not compatible with bisulfite sequencing.

 

Do you recommend a bisulfite conversion kit?

We recommend the EZ DNA Methylation-Gold™ Kit (Zymo Research, Cat # D5005)

 

What is the general efficiency of bisulfite conversion using the recommended bisulfite conversion kit?

Conversion efficiency generally exceeds 99% using the EZ DNA Methylation Kit.

Do you have protocols available for whole-genome bisulfite sequencing or reduced-representation bisulfite sequencing?

Yes, the NEXTflex™ Bisulfite-Seq Kit can be used for either WGBS or RRBS.

 

How much input DNA can be used with the NEXTflex Bisulfite-Seq Kit?

10 ng -1 µg of fragmented genomic DNA (for WGBS) or Msp1 digested genomic DNA (for RRBS) in up to 40 µL nuclease-free water.

 

How many samples can be multiplexed using the NEXTflex Bisulfite-Seq Kit? Are the indexed adapters available separately?

The methylated NEXTflex™ Bisulfite-Seq Barcodes are recommended for use with the NEXTflex Bisulfite-Seq Kit.  To offer greater flexibility, these indexed adapters are sold separately from the library preparation kits in sets of 6 or 12 unique barcodes. The 8 reaction size of the NEXTflex Bisulfite-Seq Kit (cat # 5119-01) does contain a single adapter for those customers who do not need to multiplex samples. The other barcode kits we offer are not methylated and are not compatible with bisulfite sequencing.

 

Do you recommend a bisulfite conversion kit?

We recommend the EZ DNA Methylation-Gold™ Kit (Zymo Research, Cat # D5005)

 

What is the general efficiency of bisulfite conversion using the recommended bisulfite conversion kit?

Conversion efficiency generally exceeds 99% using the EZ DNA Methylation Kit.

Do you have protocols available for whole-genome bisulfite sequencing or reduced-representation bisulfite sequencing?

Yes, the NEXTflex™ Bisulfite-Seq Kit can be used for either WGBS or RRBS.

 

How much input DNA can be used with the NEXTflex Bisulfite-Seq Kit?

10 ng -1 µg of fragmented genomic DNA (for WGBS) or Msp1 digested genomic DNA (for RRBS) in up to 40 µL nuclease-free water.

 

How many samples can be multiplexed using the NEXTflex Bisulfite-Seq Kit? Are the indexed adapters available separately?

The methylated NEXTflex™ Bisulfite-Seq Barcodes are recommended for use with the NEXTflex Bisulfite-Seq Kit.  To offer greater flexibility, these indexed adapters are sold separately from the library preparation kits in sets of 6 or 12 unique barcodes. The 8 reaction size of the NEXTflex Bisulfite-Seq Kit (cat # 5119-01) does contain a single adapter for those customers who do not need to multiplex samples. The other barcode kits we offer are not methylated and are not compatible with bisulfite sequencing.

 

Do you recommend a bisulfite conversion kit?

We recommend the EZ DNA Methylation-Gold™ Kit (Zymo Research, Cat # D5005)

 

What is the general efficiency of bisulfite conversion using the recommended bisulfite conversion kit?

Conversion efficiency generally exceeds 99% using the EZ DNA Methylation Kit.

Related Kits:

NEXTflex™ Bisulfite-Seq Barcodes

NEXTflex™ Msp1 Restriction Enzyme

 

Alternative Solutions:

NEXTflex™ Methyl-Seq 1 Kit

 

NEXTflex™ Msp1 Restriction Enzyme
NEXTflex™ Msp1 Restriction Enzyme
NEXTflex™ Msp1 Restriction Enzyme
NEXTflex™ Msp1 Restriction Enzyme
NEXTflex™ Msp1 Restriction Enzyme
NEXTflex™ Msp1 Restriction Enzyme
NEXTflex™ Msp1 Restriction Enzyme
NEXTflex™ Msp1 Restriction Enzyme