Benchmarking DNA methylation analysis of 14 alignment algorithms for whole genome bisulfite sequencing in mammals

Whole genome bisulfite sequencing (WGBS) is an essential technique for methylome studies. Although a series of tools have been developed to overcome the mapping challenges caused by bisulfite treatment, the latest available tools have not been evaluated on the performance of reads mapping as well as on bio-logical insights in multiple mammals. Herein, based on the real and simulated WGBS data of 14.77 billion reads, we undertook 936 mappings to benchmark and evaluate 14 wildly utilized alignment algorithms from reads mapping to biological interpretation in humans, cattle and pigs: Bwa-meth, BSBolt, BSMAP, Walt, Abismal, Batmeth2, Hisat_3n, Hisat_3n_repeat, Bismark-bwt2-e2e, Bismark-his2, BSSeeker2-bwt, BSSeeker2-soap2, BSSeeker2-bwt2-e2e and BSSeeker2-bwt2-local. Specifically, Bwa-meth, BSBolt, BSMAP, Bismark-bwt2-e2e and Walt exhibited higher uniquely mapped reads, mapped precision, recall and F1 score than other nine alignment algorithms, and the influences of distinct alignment algorithms on the methylomes varied considerably at the numbers and methylation levels of CpG sites, the calling of differentially methylated CpGs (DMCs) and regions (DMRs). Moreover, we reported that BSMAP showed the highest accuracy at the detection of CpG coordinates and methylation levels, the calling of DMCs, DMRs, DMR-related genes and signaling pathways. These results suggested that careful selection of algo-rithms to profile the genome-wide DNA methylation is required, and our works provided investigators with useful information on the choice of alignment algorithms to effectively improve the DNA methyla-tion detection accuracy in mammals.(c) 2022 The Author(s). Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY-NC-ND license (http://creative-commons.org/licenses/by-nc-nd/4.0/).

Automated summary

Whole genome bisulfite sequencing is crucial for methylome studies. This study evaluated the performance of 14 commonly used alignment algorithms for WGBS in multiple mammals and found that the choice of algorithm significantly impacts CpG site counts, methylation levels, and the calling of differentially methylated regions. BSMAP showed the highest accuracy in detecting CpG coordinates and methylation levels.