Elementolab/RRBSseeqer Tutorial

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Elementolab/ Elementolab/RRBSseeqer/

Got to RRBS directory

cd RRBS

Contents 1 Transform epicore methycall files into RRBSeeqer input files (.calls) 2 Detect differentially methylated CGs 3 Detect differentially methylated regions (DMR) 4 Annotate CGs 5 Draw methylation across chromosomes 6 Make BigWig DIFFERENTIAL methylation track 7 Methylation levels for annotated elements

Transform epicore methycall files into RRBSeeqer input files (.calls)

perl epicore2calls.pl methylCall_Sample_YYY.txt > methylCall_Sample_YYY.txt.calls

(optionally you can gzip the call files as most other RRBSseeqer programs can read from gzipped files)

Detect differentially methylated CGs

./RRBSseeqer_CG -rrbs1 sample.calls.gz -rrbs2 sample_control.calls.gz | sort_column.pl 1 | sort_column_alpnum.pl 0 > cgs.txt

Options:

-rrbs1		FILE (condition 1)
-rrbs2		FILE (condition 2)
-test		[fisher|chi] (Statistical test to be performed)
-minfold	FLOAT (Def: 1.25)
-fdr		FLOAT (Def: 0.2)

Detect differentially methylated regions (DMR)

perl RRBSidentifyUpDownDMR.pl --metfile=cgs.txt --dmax=250 --minmetdx=0.1 --minnumcg=5 -outfile=cgs_DMR.txt

Input must come from RRBSeeqer_CG. Parameters indicate maximum distance between diff methylated CGs (--dmax), minimum number of differentially methylated CGs for a region to be reported (--minumcg) and min total methylation dx for a region (--minmetdx), with 10% used default.

To create a bed file with all DMRs

perl DMR2bed.pl cgs_DMR.txt > cgs_DMR.bed

To create a bed file that can be uploaded to the Genome Browser, with two different tracks for hyper and hypo DMRs

perl DMR2bed2.pl cgs_DMR.txt

Annotate CGs

With CpG islands

./RRBSannotate -peakfile1 cgs.txt -peakfile2 DATA/hg19/CpGislands_annotation -showpeakdesc 1 -hasid1 0 -outfile cgs_CGI.txt

With RRBSannotate, you can just add annotation columns. Here's how to add shore annotation

./RRBSannotate -peakfile1 cgs_CGI.txt -peakfile2 DATA/hg19/CpGislands_annotation -shores 1 -ext2 1000  -hasid2 0  -showpeakdesc 1 -hasid1 0 -outfile cgs_CGI_shores.txt

With genes

./RRBSannotateGenes -rrbsfile cgs.txt -annotation DATA/hg19/refSeq -chrdata DATA/hg19.chrdata 

(you need to download gene annotation files first (e.g., hg18.tar.gz, hg19.tar.gz) from --> http://physiology.med.cornell.edu/faculty/elemento/lab/CS_files/)

Draw methylation across chromosomes

perl DrawRRBSmap2.pl --chrdata=DATA/hg19.chrdata --rrbsdata=cgs.txt

Make BigWig DIFFERENTIAL methylation track

Let's assume ben1_pca1.txt contains a differential methylation analysis generated by RRBSseeqer_CG above. To generate a BigWig track, make sure wigToBigWig is installed, and type something like this

RRBSmakeMethylDiffTrack -methfile ben1_pca1.txt -desc ben1_pca1 -type diff -bigwig 1 -chrdata hg19.chrdata -chromsizes hg19.chroms.sizes -s 25

This command will create a .bw track (binary format), with window size 50bp (2x25) that you can upload to your web server, then make visible to the UCSC browser using:

track type=bigWig name="ben1_pca1" description="ben1_pca1" bigDataUrl=http://physiology.med.cornell.edu/faculty/elemento/lab/files/ben1_pca1.wig.bw \ 
  visibility="full" maxHeightPixels="64:64:11" smoothingWindow="10" viewLimits="0:100" autoScale="off"

A sometimes faster way to visualize a BigWig track is to load it into IGV http://www.broadinstitute.org/igv/

Methylation levels for annotated elements

getMethylationLevelsForAnnotatedElements.pl --callfile=CpG_context_Sample_ln_cap_r.fastq.filtered.noadapt.gz_bismark.txt.calls_NM --headerhasid=0 \
--promlist=refSeq.new.u2000_d2000.PROMOTERS --minnumcg=2 --col=5