Package: methseq
MethSeq - Automation tool for high-throughput sequencing DNA methylation dataSupported sequencing protocols for identification of DNA methylationWGBSPico-MethylEMSeqReduced-representation bisulfite sequencing (RRBS) is not supported!Required softwarePython 3Cromwell workflow management system running in server modeDocker (optional but highly recommended to improve reproducibility)Required dataPaired-end raw sequencing FASTQ files (one file per strand)Indexed reference genome using Bismark 0.22.2 and Bowtie 2.3.5.1Used software via Docker container imagesTrimGalore version 0.6.5Bismark 0.22.2Bowtie 2.3.5.1FastQCYour compute environment may not support Docker. Install those software locally and overwrite their paths usingMethSeqcommand-line parameters.Main data processing tasksTrim and filter raw sequencing reads (TrimGalore!)Align filtered reads to methylation-aware indexed reference genome (Bismark, Bowtie 2)Deduplicate aligned reads to remove PCR-bias (Bismark)Extract DNA methylation of CpG context genome-wide (Bismark)Quality control and report tasksQuality assessment of filtered sequencing reads (FastQC)Overall report (Bismark)Expected result files for each sample, independently of sequencing protocolcpg_reportGenome-wide DNA methylation in CpG context, contains strand informationcovCoverage file, doesn't contain strand information (only CpG with coverage)bedgraphBedGraph filembias_png_1M-Bias plot of forward strand (R1)mbias_png_2M-Bias Plot of reverse strand (R2, except PicoMethyl)trim_stats_1Trimming statistics of forward strand (R1)trim_stats_2Trimming statistics of reverse strand (R2)align_statsAlignment statsnucleotide_coveragededuplicate_statsDeduplication statisticsmbias_statsM-Bias statistics (used to generate M-Bias plots)splitting_statstrim_qc_report_1QC report of filtered reads of forward strand (R1)trim_qc_report_zip_1Zipped file containing QC statistics forward strand (R1)trim_qc_report_2QC report of filtered reads of reverse strand (R2)trim_qc_report_zip_2Zipped file containing QC statistics reverse strand (R2)unmapped_qc_report_1QC report of unmapped reads of forward strand (R1)unmapped_qc_report_zip_1Zipped file containing QC statistics forward strand (R1)unmapped_qc_report_2QC report of unmapped reads of reverse strand (R2, except PicoMethyl)unmapped_qc_report_zip_2Zipped file containing QC statistics reverse strand (R2, except PicoMethyl)reportOverall reportHow to usemethseqaccel|pico|emseq--fastq/path/to/fastqs[--fastq/path/to/other_fastqs][trimmingparameters]result_dirBy default, trimming step will only trim (Illumina) adapter sequences.
Use the following trimming parameters to cut sequences after adapter removal.
It is useful to remove methylation bias saw in M-Bias plot.--five_prime_clip_R1 NremoveNbases from the beginning (5') of forward reads (R1)--three_prime_clip_R1 NremoveNbases from the end (3') of forward reads (R1)--five_prime_clip_R1 NremoveNbases from the beginning (5') of reverse reads (R2)--three_prime_clip_R1 NremoveNbases from the end (3') of reverse reads (R2)By default, trimming step will trim bases at the end (3') that have PHREAD score lower than 20.
Use--quality Nto change quality cutoff toN.By default, trimming step will filter trimmed reads that are smaller than 20 bp.
Use--length Nto change read length cutoff toN.MethSeqwillCheck if all samples are paired FASTQ filesCheck if indexed reference genome files existsGenerate inputs JSON fileWrite workflow (WDL) and JSON files to result directorySubmit WDL and JSON files to Cromwell server through its APIWait until workflow execution is completedIf success, collect workflow output files copping (or moving) them to result directoryExtra: run MultiQC onresult_dirfolder!Use casesTo process WGBS samples with trimming parameters to remove methylation bias saw in M-Bias plot.methseqwgbs\--fastq/path/to/wgbs_fastqs\--five_prime_clip_R116\--three_prime_clip_R116\--five_prime_clip_R216\--three_prime_clip_R216\/path/to/wgbs_resultTo process a single sample (EMSeq) passing path to specific file.
It is useful to runMethSeqon files that are not in the same directory.methseqemseq\--fastq_1/path/to/emseq_1.fastq.gz\--fastq_2/path/to/emseq_2.fastq.gz\/path/to/wgbs_result