NEXTFLEX® Bisulfite Library Prep Kit for Illumina® Sequencing

  • 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

  • NOVA-5119-01

    8 RXNS

  • NOVA-5119-02

    48 RXNS
Download Manual
For research use only. Not for use in diagnostic procedures.


  • NOVA-511911

    48 RXNS

  • NOVA-511912

    96 RXNS

  • NOVA-511913

    192 RXNS

RRBS & WGBS Sequencing

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.

RRBS Bisulfite-Seq Kit

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

RRBS BIsulfite-Seq Kit

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

WGBS Bisulfite-Seq

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

WGBS Bisulfite-Seq

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

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 when used with an MSP1 enzyme. 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.


  • 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


  • 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)
  • 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

Bisulfite-Seq Flowchart

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.

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.

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.