epiGBS2: an improved protocol and automated snakemake workflow for highly multiplexed reduced representation bisulfite sequencing

Abstract

epiGBS is an existing reduced representation bisulfite sequencing method to determine cytosine methylation and genetic polymorphisms de novo . Here, we present epiGBS2, an improved epiGBS laboratory protocol and user-friendly bioinformatics pipeline for a wide range of species with or without reference genome. epiGBS2 decreases costs and time investment and increases user-friendliness and reproducibility. The library protocol was adjusted to allow for a flexible choice of restriction enzymes and a double digest. Instead of fully methylated adapters, semi-methylated adapters are now used. The bioinformatics pipeline was improved in speed and integrated in the snakemake workflow management system, which now makes the pipeline easy to execute, modular, and parameter settings flexible. We also provide a detailed description of the laboratory protocol, an extensive manual of the bioinformatics pipeline, which is publicly accessible on github ( https://github.com/nioo-knaw/epiGBS2) and zenodo ( https://doi.org/10.5281/zenodo.3819996 ), and example output.

Inheritance of DNA methylation differences in the mangrove Rhizophora mangle

“…We processed the raw sequencing files using the pipeline provided by van Gurp et al (2016) as in van Moorsel et al (2019), available on https://github. Com/thomasvangurp/epiGBS, with a bug‐fix modification (i.e., described in Gawehns et al, 2020); https://github.com/MWSchmid/epiGBS_Nov_2017_fixed). Briefly, we demultiplexed, quality trimmed sequencing reads, and removed the barcode sequences, then used the processed reads for de novo reference construction.…”

Phenotypic Response to Light Versus Shade Associated with DNA Methylation Changes in Snapdragon Plants (Antirrhinum majus)

“…We used a bioinformatics pipeline integrated in the snake-make workflow called epiGBS2 [ 32 ] to remove PCR duplicates and demultiplex samples. The pipeline is available at: .…”

The methylome of Biomphalaria glabrata and other mollusks: enduring modification of epigenetic landscape and phenotypic traits by a new DNA methylation inhibitor

“…We used an existing protocol called epiGBS (van Gurp et al 2016, Gawehns et al 2020, a reduced representation bisulfite sequencing method for cost-effective exploration of DNA methylation and genetic variation designed for multiplexed high-throughput sequencing to maximize sample size while losing loci. epiGBS sequencing was performed with the snails exposed to the DNMTi that showed the most significant changes in the global 5mC % (Supplementary file 2, Figure S3).…”

“…The concentration was homogenized in all samples to 10 ng/µL in a total volume of 35 µL. epiGBS library preparation was applied as described in the step-bystep most recent protocol (Gawehns et al 2020). Paired-end sequencing (2 × 150 bp) using an Illumina NextSeq™550 instrument at the Bio-Environment NGS Platform at the University of Perpignan.…”

“…Our dot blot method and a commercial ELISA-based kit showed equivalent results. For genome-wide methylation profiling we used epi-genotyping-by-sequencing method (epiGBS) (van Gurp et al 2016, Gawehns et al 2020) and we compared the results with a previous methylation information obtained by Whole Genome Bisulfite Sequencing (WGBS) (Adema et al 2017).…”