- 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.
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.
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)
AIR™ DNA Fragmentation Kit (Bioo Scientific, Cat # 5135-01) / or / Covaris System (S2, E210)
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
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
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.