NEXTflex®  Bisulfite-Seq Barcodes

Methylated Adapters for Illumina RRBS and WGBS library prep

Catalog# Product Name Quantity US List Price
NOVA-511911 NEXTflex® Bisulfite-Seq Barcodes - 6 48 rxns $657 Buy Now
NOVA-511912 NEXTflex® Bisulfite-Seq Barcodes - 12 96 rxns $1,134 Buy Now
NOVA-511913 NEXTflex® Bisulfite-Seq Barcodes - 24 192 rxns $2,042 Buy Now


  • Up to 24 methylated adapters for multiplexing Illumina RRBS and WGBS libraries are available
  • Considerably reduce your per-sample sequencing cost by barcoded multiplexing
  • Increase your sequencing scale by pooling samples on a single flow cell
  • Compatible with bisulfite-sequencing on the Illumina sequencing platforms

The NEXTflex™ Bisulfite-Seq Barcodes are designed to be used with the NEXTflex Bisulfite-Seq Kit. Unlike other NEXTflex Barcodes, the NEXTflex Bisulfite-Seq Barcodes are methylated. The NEXTflex Bisulfite-Seq Barcodes can be used to provide flexibility and high-throughput capabilities in sequencing applications. They significantly increase scale while reducing costs by allowing the user to pool multiple library preparations in a single flow cell lane. The NEXTflex Bisulfite-Seq Barcodes kits accomplish this by using an indexed adapter with a 6 nt unique sequence. This allows for proper differentiation between samples, preventing poor reads from single base errors introduced during PCR. 

These methylated adapters can be used with single, paired-end and multiplex reads. 

Selected Publications Citing the Use of the NEXTflex Bisulfite-Seq Barcodes

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.

Urich, M. A., Nery, J. R., Lister, R., Schmitz, R. J. and Ecker, J. R. (2015) MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing. Nature Protocols 10, 475–483. doi:10.1038/nprot.2014.114.

van der Graafa, A., et al.  (2015) Rate, spectrum, and evolutionary dynamics of spontaneous epimutations. PNAS. 112:21. pg. 6676–6681. doi: 10.1073/pnas.1424254112.

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 Barcodes Kits contain 6, 12 or 24 unique barcodes, enabling the user to multiplex up to 24 samples per flow cell lane. These kits ship on dry ice.

Kit Contents

NEXTflex™ Bisulfite-Seq Adapters (25 µM)
NEXTflex™ Primer Mix (12.5 µM)


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 Kit – 8 / 48 (Cat # 5119-01, 5119-02)  
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 60 mL (Beckman Coulter Genomics, Cat # A63881)
Magnetic Stand - 96 (Ambion, Cat # AM10027) / or / similar
Heat block
2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes
Nuclease-free barrier pipette tips
1.5 mL nuclease-free microcentrifuge tubes
Low melt agarose such as Low Gelling Temperature Agarose with a melt point of  65ºC (Boston Bioproducts, Cat # P-730)
1X TAE buffer
Clean razor or scalpel
SYBR Gold (Invitrogen, Cat # S11494)
UV transilluminator or gel documentation instrument
Gel electrophoresis apparatus
Electrophoresis power supply


Sequences of NEXTflex Bisulfite-Seq Barcode Indexes - Excel / PDF

Instructions for installing NEXTflex Barcode Indices in Illumina Experiment Manager


Are the NEXTflex Bisulfite-Seq Barcodes methylated?

Yes they are methylated. Methylated barcodes are required for both WGBS and RRBS bisulfite-seq applications.


With which kits can the NEXTflex Bisulfite-Seq Barcodes be used?

The NEXTflex Bisulfite-Seq Barcodes were designed to be used with the NEXTflex Bisulfite-Seq Kit; however, they are compatible with other Illumina compatible bisulfite-seq library prep methods and most other Illumina-compatible DNA-seq applications. They are not compatible with MeDIP and MeCAP applications, as these protocols require the use of unmethylated barcodes.


At what concentration are the NEXTflex Bisulfite-Seq Barcodes supplied?

They are supplied at a 25 µM concentration.


Are the NEXTflex Bisulfite-Seq Barcodes supplied with PCR primers?

Yes they are. If you need additional PCR primers, they are available in 48, 96, 100, 192, 384 and 768 reaction sizes at a cost of $1/reaction. These are supplied at a concentration of 12.5 µM.


Are the sequences of the indexes used in the NEXTflex Bisulfite-Seq Barcodes available?

Yes. A PDF containing the indexes within the NEXTflex Bisulfite-Seq Barcodes can be downloaded here.