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[[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]<br> = overlapping samples for Illumina 450k and BSPP (GA data set) = * Aim: ** To get the CpG sites that overlap between the two methods, Illumina 450k methylation arrays and BSPP assay, then calculate the correlation coefficient (will be performed by Greg) ** To extract DNA methylation values of the CpG sites in the genes associated with the 69 predictive markets (as shown in [[Media:Table_S3.xlsx| Table_S3]]). == 35 sample lists == {| {{table}} border = 1 | width="70pt" align="center" style="background:#f0f0f0;"|'''SampleID''' | align="center" style="background:#f0f0f0;"|'''MethylFreq Corr <br>(minReadDepth=10)''' | align="center" style="background:#f0f0f0;"|'''MethylFreq Corr <br>(minReadDepth=20)''' |- | 773002||0.956||0.967 |- | 1069001||0.961||0.970 |- | 1100001||0.959||0.970 |- | 825002||0.939||0.955 |- | 880003||0.935||0.952 |- | 888003||0.938||0.956 |- | 1373001||0.932||0.951 |- | 6925001||0.932||0.953 |- | 6988001||0.845||0.880 |- | 7161001||0.932||0.952 |- | 7419001||0.883||0.915 |- | 7528001||0.966||0.976 |- | 7529001||0.936||0.954 |- | 7729002||0.887||0.917 |- | 7781002||0.752||0.761 |- | 10174001||0.890||0.922 |- | 10182001||0.828||0.843 |- | 10272001||0.962||0.972 |- | 10703001||0.851||0.870 |- | 10741001||0.893||0.921 |- | 10914001||0.864||0.903 |- | 11029001||0.849||0.884 |- | 11066001||0.919||0.942 |- | 11200001||0.902||0.928 |- | 11202001||0.885||0.917 |- | 11285001||0.932||0.952 |- | 11324001||0.969||0.977 |- | 11340001||0.919||0.940 |- | 11379001||0.929||0.950 |- | 11394002||0.938||0.956 |- | 11434001||0.891||0.922 |- | 11594001||0.927||0.945 |- | 11595001||0.961||0.970 |- | 11756001||0.970||0.977 |- | 11793001||0.966||0.974 |} '''In genome-miner''' '''Directory:''' /home/nplongth/Noi_scratch/BSPP-AMD_20111012/AMD_mappig.data_hg19/AMD-35samples BED files of 35 samples are in sub directory -> GA35samples Illumina 450k marker is in '''Illumina450kMarkers.txt''' # I generate methylFreq matrix (minRD 10, min_sample 5 and min_sample28 (80%)) ## minRD 10, min_sample 5: total number of CpG sites: 693,202 sites (GA-35samplemethyl_min5_minSTD0) ## minRD 10, min_sample 28: total number of CpG sites: 413,904 sites (GA-35samplemethyl_RD10_min28_minSTD0) # minRD 10, min_sample 5, number of sites overlapping with 450k : 61,296 sites (Illumina-450k-overlapped_GA-35samples-minRD10_minSample1.txt) # minRD 10, min_sample 28, number of sites overlapping with 450k : 37,239 sites (Illumina-450k-overlapped_GA-35samples-minRD10_minSample28.txt) These numbers showed very low overlapping of the CpG sites covered by the two assays. I asked Greg to confirm that the coordinates are reported to hg19. I also check when the coordinate is -1, -2 or +1 or -2 (change the coordinate of Illuminar markers (chr_pos), ./extract-35samplesCpG.pl GA-35samplemethyl_min5_minSTD0 <Illu-1.txt | wc -l -> 0 ./extract-35samplesCpG.pl GA-35samplemethyl_min5_minSTD0 <Illu-2.txt | wc -l -> 3,112 ? ./extract-35samplesCpG.pl GA-35samplemethyl_min5_minSTD0 < Illu+1.txt | wc -l -> 0 ./extract-35samplesCpG.pl GA-35samplemethyl_min5_minSTD0 < Illu+2.txt | wc -l -> 3,104 ? This is unlikely to get more overlapping from both -2 and +2 . However, I still need to check carefully. # check duplication among the site +2, -2 to the overlapping sites from original coordinate. awk '{print $1}' Illumina-450k-overlapped_GA-35samples-minRD10_minSample5.txt > tmp-ori (61,295 sites) ./extract-35samplesCpG.pl GA-35samplemethyl_min5_minSTD0 < Illu-2.txt | awk '{print $1}' > tmp-2 (3,112 sites) ./extract-35samplesCpG.pl GA-35samplemethyl_min5_minSTD0 < Illu+2.txt | awk '{print $1}' > tmp+2 (3,104 sies) cat tmp-ori tmp-2 | sort | uniq -d | wc -l -> 3,009 cat tmp-ori tmp-2 | sort | uniq -D | wc -l -> 6,018 cat tmp-ori tmp+2 | sort | uniq -d | wc -l ->3,016 cat tmp-ori tmp+2 | sort | uniq -D | wc -l -> 6,032 This suggests that the sites that I saw when shift -2 are just the sites that have very close position and already listed in the overlapping sites. I will ignore ~ 100 sites from each +2 and -2 since it is unlikely to be to right overlapping sites. I then look at UCLA data to see if the CpG sites covered are consistently covered in GA samples and overlapping with 450k arrays markers. # I generated matrix (minRD 10, min_sample 5: UCLA_minsample5_minSTD0 -> 691,556 sites total # number of sites overlapping with 450k: 61,273 sites # number of sites overlapping with GA data set: 649,623 (94%) == Discussion with Dr. Zhang == * 2012.07.30 == Extract the CpG position in the genes associated with the 69 predictive markers == # Download Human RefSeq from genomebrowser: http://genome.ucsc.edu/cgi-bin/hgTables -> output file: Hg19.Refseq.gz -> uncompressed -> Hg19.Refseq '''Header of Hg19.Refseq:'''<br> '''#bin'''(1) name(2) chrom(3) strand(4) txStart(5) txEnd(6) cdsStart(7) cdsEnd(8) exonCount(9) exonStarts(10) exonEnds(11) score(12) name2(13) cdsStartStat(14) cdsEndStat(15) exonFrames(16) head -10 Hg19.Refseq | sed 's/\t/\n/g' ''header'': (1)#bin (2)name (3)chrom (4)strand (5)txStart (6)txEnd (7)cdsStart (8)cdsEnd (9)exonCount (10)exonStarts (11)exonEnds (12)score (13)name2 (14)cdsStartStat (15)cdsEndStat (16)exonFrames '''To generate bed file containing Chromosome position, strart position of the gene (-2,000 bp window), end postion of genes (+2,000 bp window), and gene names'''<br> awk '{print $3"\t"$5-2000"\t"$6+2000"\t"$13}' Hg19.Refseq | sort -k1,1 -k2,2n -u > Hg19.Refseq.bed<br> ''' To fix the negative integers of start position'''<br> awk '{if($2 < 0 ) $2=0; print $0}' Hg19.Refseq.bed > Hg19.Refseq.bed.fixed<br> /home/dinh/softwares/BEDTools-Version-2.16.2/bin/bedtools intersect -wao -a GA-35samples_minRD10_min5.BED.txt -b Hg19.Refseq.bed.fixed | awk '{if ($9 ~ /1/) print $0}' > GA-35samples_minRD10_min5-Hg19Refseq2kbwindow.fixed<br> ./get_overlapped_site.pl ori.tableS3.single-genelist.txt GA-35samples_minRD10_min5-Hg19Refseq2kbwindow.fixed > GA-35samples_minRD10_min5-Hg19Refseq2kbwindow-69markers-OL<br> ./extract-35samples-69Markers.pl GA-35samplemethyl_min5_minSTD0 <GA-35samples_minRD10_min5-Hg19Refseq2kbwindow-69markers-OL| sort |uniq -u > methylData.minRD10_minsmaple5.2kbwindow.69markers-assocgenes.txt<br> ./extract-35samplesCpG.pl GA-35samplemethyl_min5_minSTD0 < tableS3-2.txt > methylData.minRD10_minsmaple5.69markers-overlap.txt<br> Rename the files and send to Greg # GA-35samples_minRD10_minsample5-2kbwindow-69markers-assocgenes.gz # methylData.minRD10_minsmaple5.2kbwindow.69markers-assocgenes.txt.gz # methylData.minRD10_minsmaple5.69markers-overlap.txt.gz
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