Dinh 2011/NOTES/2011-11-21

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5fc/5caC antibody DIP-seq[edit]

Reads mapping to repeats[edit]

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