Meth10X

Meth10X (old name: WGBSX10) is a pipeline of parallel alignment of whole genome bisulfite sequencing (WGBS) or nucleosome occupancy and DNA methylation (NOMEseq) from inputs as fastq files of multi samples (approx. 8-16 samples with more than 20X coverage for each) and generating:

• An HTML report of quality control detail metrics: percent mapped reads, methylation average of CpG and non-CpG, bisulfite conversion rate, CpG islands/shores bias, coverage bias between all sites and CpG sites, percent SNP overlapping with CpG sites, Top 5 SNPs in each sample.
• MDS plots to compare among samples
• DNA methylation distribution plots of all CpG sites, CpG island sites, CpG shore sites...
• A tsv big table of CG/CHH/CHG with filtered/unfiltered SNP
• SNP calling
• PMD, LMR, UMR, HMR calling
• Bigwig files of whole genome coverage, only CpG coverage, CpG methylation of each sample for IGV visualization of these samples

Meth10X is designed for running on cluster such as SGE/PBS. It requires 4GB of memory to maintain the running of the whole workflow.

Meth10X helps to speed up the whole workflow of high coverage WGBS as well as enhance the accurate of methylation and SNP calling.

Meth10X is built based on in-house bash/python/perl/R script, Bpipe and a collection of software packages:

• [bwa, 0.7.9a] https://github.com/lh3/bwa
• [bwa-meth, 0.10] https://github.com/brentp/bwa-meth
• [samtools, 1.4] https://github.com/samtools/
• [tabix, 1.4] https://github.com/samtools/tabix
• [qualimap, 2.2.1] http://qualimap.bioinfo.cipf.es/
• [SAMStat, 1.08] http://samstat.sourceforge.net/
• [picardtools, 2.3.0] https://github.com/broadinstitute/picard
• [bedtools, 2.22.0] https://github.com/arq5x/bedtools2
• [Bpipe, 0.9.9.2] https://github.com/ssadedin/bpipe
• [bamUtil, 1.0.7] https://github.com/statgen/bamUtil
• [MethylDackel, 0.2.0] https://github.com/dpryan79/MethylDackel
• [Biscuit, 0.2.0] https://github.com/zwdzwd/biscuit
• [MethylSeekR, 1.0] https://github.com/Bioconductor-mirror/MethylSeekR
• [Vcf-tools, 0.1.15] https://github.com/vcftools
• [fastqc, 0.11.5] http://www.bioinformatics.babraham.ac.uk/projects/fastqc/
• [trimCEGX, 1.0] https://github.com/luuloi/trim.paired.read
• [makeFullDataFrame, 1.0] https://github.com/luuloi/make.full.data.frame
• [merge_columns_multi_tsv, 1.0] https://github.com/luuloi/merge_columns_multi_tsv
• [UCSC-format-file-converter, 1.0] http://hgdownload.soe.ucsc.edu/admin/exe/linux.x86_64/
• [ggplot2] https://github.com/tidyverse/ggplot2
• [BSgenome.Hsapiens.UCSC.version] https://github.com/Przemol/BSgenome.Hsapiens.UCSC.version
• [GenomicRanges] https://github.com/Bioconductor-mirror/GenomicRanges
• [rtracklayer] https://github.com/Bioconductor-mirror/rtracklayer
• [python modules] configobj, argparse

Dependencies

• Java version >= 1.8
• All the above listed software packages, here are some commands to install python and R packages
  • [python version >= 2.7.6]
    • pip install configobj argparse
  • [R version >= 3.1.2]
    • source("https://bioconductor.org/biocLite.R")
    • biocLite(c("BSgenome.Hsapiens.UCSC.hg19", "rtracklayer", "GenomicRanges", "MethylSeekR"))
    • install.packages(c("ggplot2", "data.table", "ggfortify", "knitr"))

Installation

• git clone git@github.com:luuloi/Meth10X.git
• or
• git clone https://github.com/luuloi/Meth10X.git

Inputs require

• Paired fastq files in .gz format for each sample
• Filled sample config file, example https://github.com/luuloi/WGBSX10/blob/master/config/sample.Test_ProstateC.config
• Filled system config file, example https://github.com/luuloi/WGBSX10/blob/master/config/system.Test_ProstateC.config
• Note:
  • The fastq files are located in project/raw/LNCaP/lane1/lane1_R1.fastq.gz, project/raw/sample/lane2/lane1_R2.fastq.gz of each sample.
  • Lane and sample name should not have any dots '.' in it.

First Run [TODO]

• Indexing the human genome/or download the indexed genome before alignment
• Run the pipeline: https://github.com/luuloi/WGBSX10/blob/master/example/example.sh

  • module load phuluu/python/2.7.8

  • module load gi/java/jdk1.8.0_25

  • module load phuluu/bpipe/0.9.9.2

  • mkdir -p Test_ProstateC/raw

  • copy fastq files to Test_ProstateC/raw as following

    • cp sample1/lane1/*.fastq Test_ProstateC/raw/sample1/lane1/
    • cp sample1/lane2/*.fastq Test_ProstateC/raw/sample1/lane2/
    • cp sample2/lane1/*.fastq Test_ProstateC/raw/sample2/lane1/
    • ....

  • python "WGBS10X/pipe/run_Bpipe.py" "WGBS10X/config/sample.Test_ProstateC.config" "WGBS10X/config/system.Test_ProstateC.config"

Second Run

• module load phuluu/python/2.7.8

• module load gi/java/jdk1.8.0_25

• module load phuluu/bpipe/0.9.9.2

• python "WGBS10X/pipe/run_Bpipe.py" "WGBS10X/config/sample.Prostate.config" "WGBS10X/config/system.Prostate.config"

Outputs

• HTML report

  • Table metrics of quality control of WGBS
  • Depth vs Breadth of CpG sites
  • Overlapping of DNA methylation and SNP calling
  • PMD, UMR, LMR and HMR calling
  • Distribution of methylation
  • MDS clustering of samples

• CpG big table contain the count of C and coverage of all CpG sites in the reference genome similar to CHG and CHH big table

  • #chr	position	LNCaP.C	LNCaP.cov	PrEC_1.C	PrEC_1.cov	PrEC.C	PrEC.cov
    chr1	10469	10	11	2	12	3	14
    chr1	10471	15	15	1	14	2	12
    ...	...	...	...	...	...	...	...

• CpG big table [filtered out CpG overlapping with SNP] contain the count of C and coverage of all CpG sites in the reference genome [LNCaP having SNP at postion chr1:10471 therefore this CpG methylation value is ommitted ]

  • #chr	position	LNCaP.C	LNCaP.cov	PrEC_1.C	PrEC_1.cov	PrEC.C	PrEC.cov
    chr1	10469	10	11	2	12	3	14
    chr1	10471	0	0	1	14	2	12
    ...	...	...	...	...	...	...	...

• Output folder structure

  •