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== Testing with MONOD RRBS bam files from BisReadMapper == * Since WGBS and RRBS may cover different CGIs, the CGI selected regions by MethylPurify have this depth of coverage: samtools depth XXX.CGI.sorted.bam | awk '{sum+=$3} END { print "Sum = ", sum, " Average = ",sum/NR}' >> Sum = 41941637 Average = 2.30644 * Our RRBS data have even better coverage at CGIs, the CGI regions selected by MethylPurify for PC-T-7_2 have this depth of coverage: samtools depth PC-T-7_2.merged.CGI.sorted.bam | awk '{sum+=$3} END { print "Sum = ", sum, " Average = ",sum/NR}' >> Sum = 393749988 Average = 27.9027 * This could means that MethylPurify failed when I ran on our RRBS bam files because it couldn't understand the alignments from bisReadMapper, but requires the BSMAP alignment format (may have special instructions for MethylPurify). I will figure out the differences in BSMAP and BisReadMapper bam files. * BSMAP sam file: SRR1232303.200105 0 chr1 30557958 255 90M * 0 0 ATTCCTTTATTTTGGTGGTTGGGGTGTTTTAGGGTTTTTTTTTTAATTAATTTTTTTTTCGTTGTTTTTTTTTTTTTGTTTTATGTTTTT @CCFFFFFFHHHHJICGHHIJJJJ:DFHIJHJJJHJJJJJJHFDCDDEECEEEDDDDDDDDDDDDDDDDDDDDDDDD38ACD3:(:ADDD NM:i:3 ZS:Z:++ SRR1232303.200112 16 chr1 30559194 255 90M * 0 0 ATCCCTTCCCTTTCACAACCTACTCCGAAAACCGCTAACCTAAAAATACAGCAATCTCTACTATCCTAAAAAAACAAAATCCCAAGGAAT DDDDDDDDBDDEDDDDDDDDCACAFFFHHGIJJIJIHHJIHJJJIIGJJJJJIJGIIJJIJJIHJIHJJJJJJJJJJHHHHHFFFFFCCC NM:i:2 ZS:Z:-+ * BSMAP have extra ZS:Z field that's just the strand information. MethylPurify would fail similarly as for BisReadMapper bam files, after I removed the ZS:Z field from the BSMAP bam file. I may just need to generate the ZS:Z field to get MethylPurify to work on BisReadMapper bam files. * Encoding for ZS:Z field is as follows: ++: forward strand of Watson of reference (BSW) +-: reverse strand of Watson of reference (BSWC) -+: forward strand of Crick of reference (BSC) --: reverse strand of Crick of reference (BSCC) * Make MethylPurify think that BisReadMapper bam files are from BSMAP: Add in NM:i:0 and ZS:Z:-+ (for Crick) and ZS:Z:++ (for Watson). samtools view $f | awk '{if($2 == 0) print $0"\tNM:i:0\tZS:Z:++"; if($2 == 16) print $0"\tNM:i:0\tZS:Z:-+"}' | samtools view -bSt /media/2TB_storeA/BisRef/Hg19_plusLamba/hg19_lambda.fa.fai - > mod.$f * Run MethylPurify: python2.7 /home/ddiep/Downloads/MethylPurify-2.0-20140819/methylpurify/bin/MethylPurify -f ../mod.PC-T-7_2.merged.bam -g hg19.fa -i /home/ddiep/Downloads/MethylPurify-2.0/methylpurify/db/CGI_hg19_slop1000.bed -c 10 -s 50 -b 300 * MethylPurify outputs: ... The predicted mixing ratio is: 0.175 The number of informative bins is: 304.0 The number of informative bins is less than 400, too less for the prediction of mixing ratio, continue INFO @ Mon, 15 Sep 2014 13:09:12: Running get_methyl_profile: MixingRatio:0.175 * Results from MethylPurify: [[File:mixRatio.MethylPurify_test1.png | 600px]] [[File:informativeBins.MethylPurify_test1.png | 600px]]
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