Manipulating base quality scores enables variant calling from bisulfite sequencing alignments using conventional bayesian approaches

Abstract

Background Calling germline SNP variants from bisulfite-converted sequencing data poses a challenge for conventional software, which have no inherent capability to dissociate true polymorphisms from artificial mutations induced by the chemical treatment. Nevertheless, SNP data is desirable both for genotyping and to understand the DNA methylome in the context of the genetic background. The confounding effect of bisulfite conversion however can be conceptually resolved by observing differences in allele counts on a per-strand basis, whereby artificial mutations are reflected by non-complementary base pairs. Results Herein, we present a computational pre-processing approach for adapting sequence alignment data, thus indirectly enabling downstream analysis on a per-strand basis using conventional variant calling software such as GATK or Freebayes. In comparison to specialised tools, the method represents a marked improvement in precision-sensitivity based on high-quality, published benchmark datasets for both human and model plant variants. Conclusion The presented “double-masking” procedure represents an open source, easy-to-use method to facilitate accurate variant calling using conventional software, thus negating any dependency on specialised tools and mitigating the need to generate additional, conventional sequencing libraries alongside bisulfite sequencing experiments. The method is available at https://github.com/bio15anu/revelioand an implementation with Freebayes is available at https://github.com/EpiDiverse/SNP

Variation in DNA Methylation in Avian Nestlings Is Largely Determined by Genetic Effects

“…Therefore, this is an important source of error. As part of the epiGBS2 pipeline, this should be dealt with by the double-masking method ( Nunn et al 2022 ). This preprocessing step converts nucleotides in bisulfite context to the corresponding nucleotide in the reference genome, and nucleotides which may have arisen as a result of the bisulfite treatment are given a base quality score of 0.…”

DNA methylation shows footprints of altitude selection in the clonal plant speciesFragaria vesca

“…In our study, methylation calling was performed separately for the three sequence contexts (CG, CHG and CHH), following the methods described in Sammarco et al (25). Specifically, the EpiDiverse WGBS pipeline (https://github.com/EpiDiverse/wgbs) was used for quality control, adaptor trimming, bisulfite reads mapping and methylation calling (53). We used the most recent version of the F. vesca genome (v4.0.a2) in the mapping step (54).…”

Discarded sequencing reads uncover natural variation in pest resistance inThlaspi arvense

“…For the methylome analyses we used the EpiDiverse toolkit (79), specifically designed for large WGBS datasets. We used the WGBS pipeline (https://github.com/EpiDiverse/wgbs) for read mapping and methylation calling, retained only uniquely-mapping reads longer than 30 bp, and obtained individual-sample bedGraph files for each sequence context.…”

“…We used the WGBS pipeline (https://github.com/EpiDiverse/wgbs) for read mapping and methylation calling, retained only uniquely-mapping reads longer than 30 bp, and obtained individual-sample bedGraph files for each sequence context. We then called DMRs using the DMR pipeline (79), with a minimum coverage of 4x. We compared the 20 samples with the most and the least M. periscae and Eriysiphales loads, resulting in two sets of DMRs for each sequence context.…”