TY - JOUR T1 - Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data JF - bioRxiv DO - 10.1101/165449 SP - 165449 AU - Nelly Olova AU - Felix Krueger AU - Simon Andrews AU - David Oxley AU - Miguel R. Branco AU - Wolf Reik Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/07/18/165449.abstract N2 - Background Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing.Results We performed a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the initial and main trigger of pronounced sequencing biases, and further amplification steps lead to over-representation of these artefacts. The majority of standard library preparation methods lead to a significantly biased genomic sequence representation and a marked overestimation of both absolute and relative methylation levels, with clear implications for DNA methylation studies.Conclusions We propose that amplification-free and post-bisulfite procedures should become the gold standard for WGBS library preparation. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets. ER -