Ns126:Calendar/NOTES/2016-1-5: Difference between revisions

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*RRBS Analysis Protocol: [[File:RRBS Guide-Baraham.pdf]]
*RRBS Analysis Protocol: [[File:RRBS Guide-Baraham.pdf]]


Normal tissues from RRBS data were downloaded from the ENCODE USCS genome browser (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethylRrbs/). Raw sequence data from 16 tissues were collected (Supplementary Table). Sequences were mapped to the bisulfite-converted human genome (NCBI hg19) using Bismark [1], after trimming the first three nucleotides, using the following parameters: --bowtie2 --phred64-quals --fastq -L 20 -N 1.  
*Normal tissues from RRBS data were downloaded from the ENCODE USCS genome browser (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethylRrbs/). Raw sequence data from 16 tissues were collected (Supplementary Table). Sequences were mapped to the bisulfite-converted human genome (NCBI hg19) using Bismark [1], after trimming the first three nucleotides, using the following parameters: --bowtie2 --phred64-quals --fastq -L 20 -N 1.  


1. Krueger, F. and S.R. Andrews, Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics, 2011. 27(11): p. 1571-2.
1. Krueger, F. and S.R. Andrews, Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics, 2011. 27(11): p. 1571-2.

Revision as of 18:51, 11 January 2016

Collection RRBS Dataset From Richard Myers, HAIB (Encode Project)

Data Description

  • Normal tissues from RRBS data were downloaded from the ENCODE USCS genome browser (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethylRrbs/). Raw sequence data from 16 tissues were collected (Supplementary Table). Sequences were mapped to the bisulfite-converted human genome (NCBI hg19) using Bismark [1], after trimming the first three nucleotides, using the following parameters: --bowtie2 --phred64-quals --fastq -L 20 -N 1.

1. Krueger, F. and S.R. Andrews, Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics, 2011. 27(11): p. 1571-2.

Fastq Download

xargs -n 1 curl -O -L < haib.download.files.txt

Check Phred Score

perl ~/bin/checkphred.pl *fastq

Fastq Quality Control

# trim_galore --phred64 --fastqc --illumina --non_directional --rrbs *.fastq
trim_galore --phred64 --fastqc --illumina --rrbs *.fastq

Fastq Alignment

bismark --bowtie2 --phred64-quals --fastq -L 30 -N 1 /home/shg047/db/aligndb/hg19/bismark -1 ENCFF000MLM_trimmed.fq -o ../bam

Methylation haploinfo

bismark_methylation_extractor \
--single-end \
--bedGraph \
--buffer_size 2G \
--remove_spaces \
--zero_based \
--merge_non_CpG \
--comprehensive \
--output ../methyfreq \
ENCFF000MLM_trimmed.fq_bismark_bt2.bam
bismark_methylation_extractor --single-end --bedGraph --buffer_size 2G --remove_spaces --zero_based --merge_non_CpG --comprehensive --output ../methyfreq  ENCFF000MLM_trimmed.fq_bismark_bt2.bam

Methylfreq Data Output

  • Bismark Run Perl Beginning Script
#/usr/bin/perl
use strict;
use Cwd;
my $dir=getcwd;
chdir $dir;
my @file=glob("*.fastq");
foreach my $file(@file){
open OUT,">$file.bismark.sh";
print OUT "cd $dir\n";
chomp(my $phredcheck=`perl /home/shg047/bin/checkphred.pl $file`);
my ($phred)=split /\s+/,$phredcheck;
my $phred="--phred$phred";
print OUT "trim_galore $phred --fastqc --illumina --non_directional --rrbs $file\n";
print OUT "bismark --bowtie2 --phred64-quals --fastq -L 30 -N 1 /home/shg047/db/aligndb/hg19/bismark -1 $file\_qual_trimmed.fastq -o ../bam\n";
print OUT "bismark_methylation_extractor --single-end --bedGraph --buffer_size 2G --remove_spaces --zero_based --merge_non_CpG --comprehensive --  output ../me
}

Methylation Haplotype Load Matrix

Evaluation of the data quality

Merge with our RRBS data