2012/01/01 by Aniruddha Chatterjee, Euan J. Rodger, Peter A. Stockwell +2 · 1 citation
Biochemistry, Genetics and Molecular Biology · #Biology #Bisulfite #Bisulfite sequencing #Computational biology #Computer science #DNA #DNA methylation #DNA sequencer #DNA sequencing #Deep sequencing #Epigenetics and DNA Methylation #Gene #Genetics #Genome #Illumina dye sequencing #Multiplexing #Pipeline (software) #Protocol (science) #RNA modifications and cancer #Single-cell and spatial transcriptomics #Telecommunications
paper · pdf · doi:10.1155/2012/741542
openalex publication_date 2012/01/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/27
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.