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Journal of Biomedicine and Biotechnology
Volume 2012 (2012), Article ID 741542, 8 pages
Methodology Report

Technical Considerations for Reduced Representation Bisulfite Sequencing with Multiplexed Libraries

1Department of Pathology, Dunedin School of Medicine, University of Otago, 270 Great King Street, Dunedin 9054, New Zealand
2National Research Centre for Growth and Development, 2-6 Park Avenue, Grafton, Auckland 1142, New Zealand
3Department of Biochemistry, University of Otago, 710 Cumberland Street, Dunedin 9054, New Zealand

Received 19 June 2012; Accepted 18 September 2012

Academic Editor: Wolfgang Arthur Schulz

Copyright © 2012 Aniruddha Chatterjee et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Reduced representation bisulfite sequencing (RRBS), which couples bisulfite conversion and next generation sequencing, is an innovative method that specifically enriches genomic regions with a high density of potential methylation sites and enables investigation of DNA methylation at single-nucleotide resolution. Recent advances in the Illumina DNA sample preparation protocol and sequencing technology have vastly improved sequencing throughput capacity. Although the new Illumina technology is now widely used, the unique challenges associated with multiplexed RRBS libraries on this platform have not been previously described. We have made modifications to the RRBS library preparation protocol to sequence multiplexed libraries on a single flow cell lane of the Illumina HiSeq 2000. Furthermore, our analysis incorporates a bioinformatics pipeline specifically designed to process bisulfite-converted sequencing reads and evaluate the output and quality of the sequencing data generated from the multiplexed libraries. We obtained an average of 42 million paired-end reads per sample for each flow-cell lane, with a high unique mapping efficiency to the reference human genome. Here we provide a roadmap of modifications, strategies, and trouble shooting approaches we implemented to optimize sequencing of multiplexed libraries on an a RRBS background.