NEXTFLEX® Bisulfite-Seq Barcodes

  • 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
For research use only. Not for use in diagnostic procedures.

  • NOVA-511911


    6 BARCODES | 48 RXNS

  • NOVA-511912


    12 BARCODES | 96 RXNS

  • NOVA-511913


    24 BARCODES | 192 RXNS

Pool Multiple Library Preparations in a Single Flow Cell Lane

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.


  • NEXTFLEX® Bisulfite-Seq Adapters (25 µM)
  • NEXTFLEX® Primer Mix (12.5 µM)





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.

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 bisulfite-seq library prep methods and most other DNA-seq applications compatible with Illumina® sequencing platforms. 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.

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.