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

From ZhangLabWiki
Jump to navigation Jump to search
>Shicheng
>Shicheng
 
(21 intermediate revisions by the same user not shown)
Line 6: Line 6:
*RRBS Protocol: [[File:Myers Lab RRBS Protocol 6-18-2010.pdf]]
*RRBS Protocol: [[File:Myers Lab RRBS Protocol 6-18-2010.pdf]]
*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 [1]. Raw sequence data from 16 tissues were collected (Supplementary Table). Sequences were mapped to the bisulfite-converted human genome (NCBI hg19) using Bismark [2], after trimming the first three nucleotides, using the following parameters: --bowtie2 --phred64-quals --fastq -L 20 -N 1.
*1.RRBS and ENCODE, http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethylRrbs/.
*2.Krueger, F. and S.R. Andrews, Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics, 2011. 27(11): p. 1571-2.
* Main important tissues were selected and merge to prediction model
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''ENCFF000LWX'''
| align="center" style="background:#f0f0f0;"|'''RRBS'''
| align="center" style="background:#f0f0f0;"|'''uterus'''
| align="center" style="background:#f0f0f0;"|'''adult'''
| align="center" style="background:#f0f0f0;"|'''female'''
| align="center" style="background:#f0f0f0;"|'''44 year'''
| align="center" style="background:#f0f0f0;"|'''36bp'''
| align="center" style="background:#f0f0f0;"|'''Illumina Genome Analyzer IIx'''
|-
| ENCFF000LXB||RRBS||uterus||adult||female||44 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LVO||RRBS||lung||adult||female||83 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LVR||RRBS||lung||adult||female||83 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LVA||RRBS||kidney||adult||female||83 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LVB||RRBS||kidney||adult||female||83 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LUT||RRBS||breast||adult||female||21 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LUV||RRBS||breast||adult||female||21 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LUQ||RRBS||brain||adult||male||66 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LUU||RRBS||brain||adult||male||66 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LWS||RRBS||stomach||adult||female||83 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LWW||RRBS||stomach||adult||female||83 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LVU||RRBS||pancreas||adult||male||71 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LVW||RRBS||pancreas||adult||male||71 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LWO||RRBS||zone of skin||adult||female||83 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LWP||RRBS||zone of skin||adult||female||83 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LVI||RRBS||mononuclear cell||adult||unknown||54 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LVK||RRBS||mononuclear cell||adult||unknown||54 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LVJ||RRBS||liver||adult||female||83 year||36bp||Illumina Genome Analyzer IIx
|-
| ENCFF000LVN||RRBS||liver||adult||female||83 year||36bp||Illumina Genome Analyzer IIx
|-
|
|}


===Fastq Download===
===Fastq Download===
*Fastq donwload address: [[File:Haib.download.files.txt]]
*Fastq donwload address: [[File:Haib.download.files.txt]]
* Sample Information:[[excel]]
  xargs -n 1 curl -O -L < haib.download.files.txt
  xargs -n 1 curl -O -L < haib.download.files.txt


Line 15: Line 72:


===Fastq Quality Control===
===Fastq Quality Control===
 
*trim_galore can used gz compressed fastq file as the input (so as the bismark)
  # trim_galore --phred64 --fastqc --illumina --non_directional --rrbs *.fastq
  # trim_galore --phred64 --fastqc --illumina --non_directional --rrbs *.fastq
  trim_galore --phred64 --fastqc --illumina --rrbs *.fastq
  trim_galore --phred64 --fastqc --illumina --rrbs *.fastq


===Fastq Alignment===
===Fastq Alignment===
 
* bismark can used gz compressed fastq file as the input (so as the trim_galore)
  bismark --bowtie2 --phred64-quals --fastq -L 30 -N 1 /home/shg047/db/aligndb/hg19/bismark -1 ENCFF000LUN.fastq_qual_trimmed.fastq -o ../bam
  bismark --bowtie2 --phred64-quals --fastq -L 30 -N 1 /home/shg047/db/aligndb/hg19/bismark -1 ENCFF000MLM_trimmed.fq -o ../bam


===Methylation haploinfo===
===Methylation haploinfo===
Line 34: Line 91:
  --comprehensive \
  --comprehensive \
  --output ../methyfreq \
  --output ../methyfreq \
  ENCFF000LUN.fastq_qual_trimmed.fastq.bam
  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  ENCFF000LUN.fastq_qual_trimmed.fastq.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===
===Methylfreq Data Output===
 
* Bismark Run Perl Beginning Script
  #/usr/bin/perl
  #/usr/bin/perl
  use strict;
  use strict;
Line 56: Line 113:
  print OUT "bismark_methylation_extractor --single-end --bedGraph --buffer_size 2G --remove_spaces --zero_based --merge_non_CpG --comprehensive --  output ../me
  print OUT "bismark_methylation_extractor --single-end --bedGraph --buffer_size 2G --remove_spaces --zero_based --merge_non_CpG --comprehensive --  output ../me
  }
  }
*Bismark Alignment Information Collection Script
** Mapping efficency: [[File:BismarkAlignment.txt]]


===Methylation Haplotype Load Matrix===
===Methylation Haplotype Load Matrix===
* methylation haplotype load for Haib data were save in TSCC
  /home/shg047/oasis/Haib/haploinfo/methHapLoad.matrix.txt
===Methylation frequency Load Matrix===
* merge zero-based methylation cov files.
#!/bin/csh
#PBS -q pdafm
#PBS -l nodes=1:ppn=1
#PBS -l walltime=1:00:00
#PBS -V
#PBS -M shihcheng.guo@gmail.com
#PBS -m abe
#PBS -A k4zhang-group
perl methylfreq2matrix.pl


===Evaluation of the data quality===
===Evaluation of the data quality===
* cluster analysis


===Merge with our RRBS data===
===Merge with our RRBS data===
* data were saved in
/home/shg047/oasis/monod/2016/monod.merge.wgbs.rrbs.txt

Latest revision as of 21:54, 19 January 2016

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

Data Description[edit]

  • Normal tissues from RRBS data were downloaded from the ENCODE USCS genome browser [1]. Raw sequence data from 16 tissues were collected (Supplementary Table). Sequences were mapped to the bisulfite-converted human genome (NCBI hg19) using Bismark [2], after trimming the first three nucleotides, using the following parameters: --bowtie2 --phred64-quals --fastq -L 20 -N 1.
  • 1.RRBS and ENCODE, http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeHaibMethylRrbs/.
  • 2.Krueger, F. and S.R. Andrews, Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics, 2011. 27(11): p. 1571-2.
  • Main important tissues were selected and merge to prediction model
ENCFF000LWX RRBS uterus adult female 44 year 36bp Illumina Genome Analyzer IIx
ENCFF000LXB RRBS uterus adult female 44 year 36bp Illumina Genome Analyzer IIx
ENCFF000LVO RRBS lung adult female 83 year 36bp Illumina Genome Analyzer IIx
ENCFF000LVR RRBS lung adult female 83 year 36bp Illumina Genome Analyzer IIx
ENCFF000LVA RRBS kidney adult female 83 year 36bp Illumina Genome Analyzer IIx
ENCFF000LVB RRBS kidney adult female 83 year 36bp Illumina Genome Analyzer IIx
ENCFF000LUT RRBS breast adult female 21 year 36bp Illumina Genome Analyzer IIx
ENCFF000LUV RRBS breast adult female 21 year 36bp Illumina Genome Analyzer IIx
ENCFF000LUQ RRBS brain adult male 66 year 36bp Illumina Genome Analyzer IIx
ENCFF000LUU RRBS brain adult male 66 year 36bp Illumina Genome Analyzer IIx
ENCFF000LWS RRBS stomach adult female 83 year 36bp Illumina Genome Analyzer IIx
ENCFF000LWW RRBS stomach adult female 83 year 36bp Illumina Genome Analyzer IIx
ENCFF000LVU RRBS pancreas adult male 71 year 36bp Illumina Genome Analyzer IIx
ENCFF000LVW RRBS pancreas adult male 71 year 36bp Illumina Genome Analyzer IIx
ENCFF000LWO RRBS zone of skin adult female 83 year 36bp Illumina Genome Analyzer IIx
ENCFF000LWP RRBS zone of skin adult female 83 year 36bp Illumina Genome Analyzer IIx
ENCFF000LVI RRBS mononuclear cell adult unknown 54 year 36bp Illumina Genome Analyzer IIx
ENCFF000LVK RRBS mononuclear cell adult unknown 54 year 36bp Illumina Genome Analyzer IIx
ENCFF000LVJ RRBS liver adult female 83 year 36bp Illumina Genome Analyzer IIx
ENCFF000LVN RRBS liver adult female 83 year 36bp Illumina Genome Analyzer IIx

Fastq Download[edit]

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

Check Phred Score[edit]

perl ~/bin/checkphred.pl *fastq

Fastq Quality Control[edit]

  • trim_galore can used gz compressed fastq file as the input (so as the bismark)
# trim_galore --phred64 --fastqc --illumina --non_directional --rrbs *.fastq
trim_galore --phred64 --fastqc --illumina --rrbs *.fastq

Fastq Alignment[edit]

  • bismark can used gz compressed fastq file as the input (so as the trim_galore)
bismark --bowtie2 --phred64-quals --fastq -L 30 -N 1 /home/shg047/db/aligndb/hg19/bismark -1 ENCFF000MLM_trimmed.fq -o ../bam

Methylation haploinfo[edit]

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[edit]

  • 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[edit]

  • methylation haplotype load for Haib data were save in TSCC
 /home/shg047/oasis/Haib/haploinfo/methHapLoad.matrix.txt


Methylation frequency Load Matrix[edit]

  • merge zero-based methylation cov files.
#!/bin/csh
#PBS -q pdafm
#PBS -l nodes=1:ppn=1
#PBS -l walltime=1:00:00
#PBS -V
#PBS -M shihcheng.guo@gmail.com
#PBS -m abe
#PBS -A k4zhang-group
perl methylfreq2matrix.pl

Evaluation of the data quality[edit]

  • cluster analysis

Merge with our RRBS data[edit]

  • data were saved in
/home/shg047/oasis/monod/2016/monod.merge.wgbs.rrbs.txt