Dinh 2011/NOTES/2011-11-21
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5fc/5caC antibody DIP-seq[edit]
Reads mapping to repeats[edit]
- continued from Dinh 2011/NOTES/2011-11-14
- Unix sort of repeatTags and then using samtool's rmdup
awk '{if($1 !~ /SQ/ && $1 !~ /PN/) print $0}' 5fC.repeatTags.sam | sort -k3,3 -k4,4n | samtools view -uSt ../../mm9Annotations-iGenomeUCSC/BWAIndex/genome.fai - | samtools rmdup - rmdup.tmp samtools view rmdup.tmp > 5fC.repeatTags.rmdup.sam awk '{if($1 !~ /SQ/ && $1 !~ /PN/) print $0}' 5caC.repeatTags.sam | sort -k3,3 -k4,4n | samtools view -uSt ../../mm9Annotations-iGenomeUCSC/BWAIndex/genome.fai - | samtools rmdup - rmdup.tmp samtools view rmdup.tmp > 5caC.repeatTags.rmdup.sam awk '{if($1 !~ /SQ/ && $1 !~ /PN/) print $0}' IgG.repeatTags.sam | sort -k3,3 -k4,4n | samtools view -uSt ../../mm9Annotations-iGenomeUCSC/BWAIndex/genome.fai - | samtools rmdup - rmdup.tmp samtools view rmdup.tmp > IgG.repeatTags.rmdup.sam awk '{if($1 !~ /SQ/ && $1 !~ /PN/) print $0}' Input.repeatTags.sam | sort -k3,3 -k4,4n | samtools view -uSt ../../mm9Annotations-iGenomeUCSC/BWAIndex/genome.fai - | samtools rmdup - rmdup.tmp samtools view rmdup.tmp > Input.repeatTags.rmdup.sam
- Number of tags after removing clonal tags (remember these are 'tags' and not reads because there can be multiple entries for the same reads)
19137939 5caC.repeatTags.rmdup.sam 4854260 5fC.repeatTags.rmdup.sam 4078937 IgG.repeatTags.rmdup.sam 6198027 Input.repeatTags.rmdup.sam
- Random sorting and rebalancing the tags (macs14 will do some additional filtering, but at least the number of tags will be close):
sort -R 5caC.repeatTags.rmdup.sam | head -4078937 > sampled.5caC.repeatTags.rmdup.sam sort -R 5fC.repeatTags.rmdup.sam | head -4078937 > sampled.5fC.repeatTags.rmdup.sam sort -R Input.repeatTags.rmdup.sam | head -4078937 > sampled.Input.repeatTags.rmdup.sam
- Running macs14 in parallel:
nohup macs14 -t sampled.5caC.repeatTags.rmdup.sam -c sampled.Input.repeatTags.rmdup.sam -f SAM -g mm -n 5caC-vsInput -w --call-subpeaks > macs14_5caCvsInput_balanced & nohup macs14 -t sampled.5fC.repeatTags.rmdup.sam -c IgG.repeatTags.rmdup.sam -f SAM -g mm -n 5fC-vsIgG -w --call-subpeaks > macs14_5fCvsIgG_balanced & nohup macs14 -t sampled.5caC.repeatTags.rmdup.sam -c IgG.repeatTags.rmdup.sam -f SAM -g mm -n 5caC-vsIgG -w --call-subpeaks > macs14_5caCvsIgG_balanced & nohup macs14 -t sampled.5fC.repeatTags.rmdup.sam -c sampled.Input.repeatTags.rmdup.sam -f SAM -g mm -n 5fC-vsInput -w --call-subpeaks > macs14_5fCvsInput_balanced &
- Somehow, macs14 is still filtered out a lot of reads from each file, so I checked the *.rmdup.sam files and found that some clonal reads were not removed by rmdup:
Clonal tags which remained in rmdup: HWI-EAS540-C:4:10:19388:9721#0:alt1 16 chr1 3005965 0 40M * 0 0 CAAAACAACCCTGAGATTCCACTTCACTCCAGTGAGAATG HHHHHHHHHGGGGGGGGFGGHHEHHHHHHHGGDGGA;;>@ NM:i:0 HWI-EAS540-C:4:50:7522:17537#0:alt1 16 chr1 3005965 0 40M * 0 0 CAAAACAACCCTGAGATTCCACTTCACTCCAGTGAGAATG IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHIIGE?DBE NM:i:0 HWI-EAS540-C:4:58:3882:4518#0:alt1 16 chr1 3022945 0 40M * 0 0 AAAAACAATCTACAGATTCAATGAAATCCCCATCAAAATT IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIGGGGG NM:i:0 HWI-EAS540-C:4:74:3471:6745#0:alt1 16 chr1 3022945 0 40M * 0 0 AAAAACAATCTACAGATTCAATGAAATCCCCATCAAAATT IIIIIIIIIIIIIIIIHIIIIIIHIIIIIIIIIIIGGGGG NM:i:0