Improve-RRBS: a novel tool to correct the 3' trimming of reduced representation sequencing reads.

Motivation: Reduced Representation Bisulfite Sequencing (RRBS) is a popular approach to determine DNA methylation of the CpG-rich regions of the genome. However, we observed that false positive differentially methylated sites (DMS) are also identified using the standard computational analysis. Results: During RRBS library preparation the MspI digested DNA undergo end-repair by a cytosine at the 3' end of the fragments. After sequencing, Trim Galore cuts these end-repaired nucleotides. However, Trim Galore fails to detect end-repair when it overlaps with the 3' end of the sequencing reads. We found that these non-trimmed cytosines bias methylation calling, thus, can identify DMS erroneously. To circumvent this problem, we developed improve-RRBS, which efficiently identifies and hides these cytosines from methylation calling with a false positive rate of maximum 0.5%. To test improve-RRBS, we investigated four datasets from four laboratories and two different species. We found non-trimmed 3' cytosines in all datasets analyzed and as much as >50% of false positive DMS under certain conditions. By applying improve-RRBS, these DMS completely disappeared from all comparisons. Availability and implementation: Improve-RRBS is a freely available python package https://pypi.org/project/iRRBS/ or https://github.com/fothia/improve-RRBS to be implemented in RRBS pipelines.