DNA methylation is an important epigenetic control that plays a major role in gene expression, splicing, and genomic imprinting. Current bisulfite conversion, coupled with next-generation sequencing (NGS)-based methods, e.g., whole-genome bisulfite sequencing (WGBS) and reduced representation bisulfite sequencing (RRBS), are able to profile genome-wide DNA methylation at single base-pair resolution. Subsequent analysis of NGS data proceeds with the alignment of the bisulfite reads. However, sodium bisulfite treatment converts each unmethylated cytosine (C) to uracil, so an aligner needs to allow a thymine (T) in the read to match to a C in the reference genome when an unmethylated C occurs.
Since the early 2010s, various algorithms have been proposed to accomplish such alignments; among them, BS-Seeker, Bismark, and BSMAP were the earliest developed and are the most commonly used [1,2,3]. The former two employ an “in silico bisulfite conversion” strategy that results in a three-letter genome, where all the Cs in both the reads and the reference are converted to Ts prior to alignment. In contrast, BSMAP aligns with a wildcard approach. The recent version of BS-Seeker, BS-Seeker2, is arguably one of the most versatile bisulfite aligners [4]; it can map reads from both WGBS and RRBS, allows gapped local alignment, and supports a suite of traditional DNA aligners. Additionally, the advances in high-throughput sequencing technologies in recent years had significantly lowered sequencing cost and affordability. Current bisulfite aligners need to be updated to process this out-burst of information in a timely manner.
Here, we introduce BS-Seeker3, an extensively improved and optimized implementation of BS-Seeker2 that leverages the available computational power of a standard bioinformatics lab. BS-Seeker3 adopts all alignment features of BS-Seeker2, some of them include the support for local and gapped alignment, RRBS mapping, and built-in adapter trimming [4].
BS-Seeker3 also incorporates a series of new features to achieve significantly faster speed and better accuracy compared to other available bisulfite aligners. It is 1.5X faster than BSMAP, 10X faster than Bismark and Brat-nova, and maps twice as many reads as either of those aligners [2, 3, 5]. BS-Seeker3 also offers downstream analysis of bisulfite read data to further investigate bisulfite conversion efficiency and to visualize the methylation pattern after alignment. It is also well integrated with downstream methylation analyzer MethGo to provide a variety of genomic and epigenomic analyses [6].