Kun:LabNotes/MONOD/2014-8-12

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RRBS data analysis (continued)

1. Compile a list of RRBS targets

  • Take all the RRBS data that we generated from primary tumor samples, concatenate all the methylFreq files, and generate a single BED file.
 cat 6-T-1_1.methylFreq 6-T-1_2.methylFreq 6-T-2_1.methylFreq 6-T-2_2.methylFreq 6-T-3_1.methylFreq 6-T-3_2.methylFreq 6-T-4_1.methylFreq 6-T-4_2.methylFreq 6-T-5_1.methylFreq 6-T-5_2.methylFreq 7-T-1_1.methylFreq 7-T-1_2.methylFreq 7-T-2_1.methylFreq 7-T-2_2.methylFreq 7-T-3_1.methylFreq 7-T-3_2.methylFreq 7-T-4_1.methylFreq 7-T-4_2.methylFreq 7-T-5_1.methylFreq 7-T-5_2.methylFreq CTT-FFPE-100ng_1.methylFreq CTT-FFPE-100ng_2.methylFreq CTT-FFPE-5ng_1.methylFreq CTT-FFPE-5ng_2.methylFreq CTT-frozen-100ng_1.methylFreq CTT-frozen-100ng_2.methylFreq CTT-frozen-5ng_1.methylFreq CTT-frozen-5ng_2.methylFreq PC-T-1_1.methylFreq PC-T-1_2.methylFreq PC-T-2_1.methylFreq PC-T-2_2.methylFreq PC-T-4_1.methylFreq PC-T-4_2.methylFreq PC-T-6_1.methylFreq PC-T-6_2.methylFreq PC-T-7_1.methylFreq PC-T-7_2.methylFreq |  /home/dinh/scripts/methylFreq2BED.pl  20 > /home/kunzhang/CpgMIP/Data/MONOD/MONOD_primary_tumor_RRBS_combined.BED.txt
 cat MONOD_primary_tumor_RRBS_combined.BED.txt | sort -k1,1 -k2,2n > MONOD_primary_tumor_RRBS_combined.sorted.BED.txt  
  • Group the sites covered into clusters:
 ./bed2Clusters.pl MONOD_primary_tumor_RRBS_combined.sorted.BED.txt > MONOD_primary_tumor_RRBS_targets.BED.txt
  • A total of 198,439 regions in the autosomes, with a total size of 48.8Mb.

2. Check the total read depth for CpG sites within the RRBS targets

 cat 6-P-10.methylFreq | /home/dinh/scripts/methylFreq2BED.pl 1 |  /home/kunzhang/softwares/bedtools-2.17.0/bin/bedtools intersect -wa -a - -b /home/kunzhang/CpgMIP/Data/MONOD/MONOD_primary_tumor_RRBS_targets.BED.txt | /home/kunzhang/CpgMIP/Data/MONOD/bed_total_RD.pl
 cat 6-P-10.methylFreq | /home/dinh/scripts/methylFreq2BED.pl 1 | /home/kunzhang/CpgMIP/Data/MONOD/bed_total_RD.pl
 
                On-target read depth   Total CpG read depth   % on-target
 6-P-10             15,965,319               20,663,932          77.3%
 6-P-1              10,145,982               13,357,081          76.0%
 6-T-1_1            42,122,819               51,404,477          81.9%
 6-T-2_1            39,727,871               47,770,689          83.2%
 CTT-frozen-100ng_1 50,274,814               55,387,277          90.8%
 CTT-frozen-5ng_1   14,303,236               15,310,475          93.4%