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README.md

Methylome v1.3

Methylome workflow , which agree to FAIR principles , was built in Nexflow dsl2 language, with singularity container for used softwares, optimized in terms of computing resources (cpu, memory) for fish genomes studies, and its use on a informatic farm with a slurm scheduler.

  • Quality and adapter trimming from paired-end reads was carried out using Trim Galore [1].
  • Trimmed reads mapping, remove duplicates (if required), and methylation calling was achieved with Bismark [2].
  • BigWig files were obtained with bedGraphToBigWig [3].

Install methylome flow and build singularity image

Clone methylome git and build local singularity image (with system admin rights) based on the provided singularity definition file.

git clone https://forgemia.inra.fr/lpgp/methylome.git
sudo singularity build ./methylome/singularity/methylome.sif ./methylome/singularity/methylome.def

Usage examples

design.csv file must have ID, R1 and R2 header and write with comma separator.

ID R1 R2
A /path/to/targetA_R1.fa.gz /path/to/targetA_R2.fa.gz
B /path/to/targetB_R1.fa.gz /path/to/targetB_R2.fa.gz
C /path/to/targetC_R1.fa.gz /path/to/targetC_R2.fa.gz

RRBS

#!/bin/bash
#SBATCH -J methylation
#SBATCH -p unlimitq
module load containers/singularity/3.9.9
module load bioinfo/Nextflow/21.10.6
nextflow run /work/project/lpgp/Nextflow/methylome/ \
-profile slurm \
--input "${PWD}/design.csv" \
--genome "genome.dna.toplevel.fa" \
--rrbs \
--ignore 2 \
--ignore_3prime 2 \
--ignore_r2 2 \
--ignore_3prime_r2 2 \
--scaffolds \
--buffer_size '50G' \
--meth_ratio_matrix \
--bigwig \
--out_dir "${PWD}/results"

RRBS for bisulfite conversion computation

#!/bin/bash
#SBATCH -J methylation
#SBATCH -p unlimitq
module load containers/singularity/3.9.9
module load bioinfo/Nextflow/21.10.6
nextflow run /work/project/lpgp/Nextflow/methylome/ \
-profile slurm \
--input "${PWD}/design.csv" \
--genome "${baseDir}/spikes_templates/diagenode/diagenode_spikes.fa" \
--rrbs \
--ignore 2 \
--ignore_3prime 2 \
--ignore_r2 2 \
--ignore_3prime_r2 2 \
--CX \
--out_dir "${PWD}/spikes"

WGBS

#!/bin/bash
#SBATCH -J methylation
#SBATCH -p unlimitq
module load containers/singularity/3.9.9
module load bioinfo/Nextflow/21.10.6
nextflow run /work/project/lpgp/Nextflow/methylome/ \
-profile slurm \
--input "${PWD}/design.csv" \
--genome "genome.dna.toplevel.fa" \
--three_prime_clip_r1 10 \
--clip_r2 10 \
--scaffolds \
--buffer_size '50G' \
--meth_ratio_matrix \
--bigwig \
--out_dir "${PWD}/results"

Defaults parameters

Please refer to Trim Galore and Bismark for complete arguments explanation.

# fastq input
input = false

# genome index
genome = false
bismarkIndex = false
keep_index = false

# fastqc
skip_fastqc = false

# trimming
skip_trimming = false
clip_r1 = 0
clip_r2 = 0
three_prime_clip_r1 = 0
three_prime_clip_r2 = 0
rrbs = false
keep_trimmed = false

# Bimark alignment
local = false
score_min = 0.2

# bismark_methylation_extractor
ignore = 0
ignore_3prime = 0
ignore_r2 = 0
ignore_3prime_r2 = 0
CX = false
scaffolds = false
buffer_size = false

# merge methyation ratio
meth_ratio_matrix=false

# bigwig
bigwig=false

# save directory
out_dir = "${PWD}/results"

References

  1. Krueger F, Galore T. A wrapper tool around cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files [Internet]. Available from: http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/
  2. Krueger F, Andrews SR. Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics. 2011;27:1571–2.
  3. Kent WJ, Zweig AS, Barber G, Hinrichs AS, Karolchik D. BigWig and BigBed: enabling browsing of large distributed datasets. Bioinformatics. 2010;26:2204–7.