Matt:LabNotes/2015-4-27
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Checking RNA-Seq Read Coverage of Probe Targets[edit]
- Probes were already filtered for those with low coverage in bulk tissue RNA-Seq with all Brodmann Areas summed together
- Still, we can check just BA8 to see if that explains lack of detection of certain genes
Create BED File for Probe Targets[edit]
- CA12kNov2014_Probelist2BED.pl
- Made start and end positions +/- 25bp from target position
- OutputFile_0gap_contig_final.full_info.V4.txt > CA12k_Nov2014_V4.bed
- OutputFile_0gap_final.full_info.V7.txt > CA12k_Nov2014_V7.bed
- Convert from hg38 reference positions to hg19 reference (which RNA-Seq data was mapped to) using UCSC LiftOver
- CA12k_Nov2014_V4.bed > CA12k_Nov2014_V4_hg19.bed
- CA12k_Nov2014_V7.bed > CA12k_Nov2014_V7_hg19.bed
Bedtools Coverage with RNA-Seq Data[edit]
- Workspace
- genome-miner:/home/mzcai/CA12kNov2014_V4_CaptureAnalysis
- genome-miner:/home/mzcai/CA12kNov2014_V7_CaptureAnalysis
- RNA-Seq data from: /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR$
- RL-BA8-sec9-t-N701-15May14_S1_mapped/RL-BA8-sec9-t-N701-15May14_S1_Aligned.sorted.bam
- 59,038,666 reads
- RL-BA8-sec9-n-N702-15May14_S2_mapped/RL-BA8-sec9-n-N702-15May14_S2_Aligned.sorted.bam
- 45,222,268 reads
- RL-BA8-sec9-t-N701-15May14_S1_mapped/RL-BA8-sec9-t-N701-15May14_S1_Aligned.sorted.bam
- Commands
/home/mzcai/softwares/bedtools-2.20.1/bin/coverageBed -abam /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/RL-BA8-sec9-t-N701-15May14_S1_mapped/RL-BA8-sec9-t-N701-15May14_S1_Aligned.sorted.bam -b CA12k_Nov2014_V4_hg19.bed > coverage.BA8-t.V4.txt & /home/mzcai/softwares/bedtools-2.20.1/bin/coverageBed -abam /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/RL-BA8-sec9-n-N702-15May14_S2_mapped/RL-BA8-sec9-n-N702-15May14_S2_Aligned.sorted.bam -b CA12k_Nov2014_V4_hg19.bed > coverage.BA8-n.V4.txt & /home/mzcai/softwares/bedtools-2.20.1/bin/coverageBed -counts -abam /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/RL-BA8-sec9-t-N701-15May14_S1_mapped/RL-BA8-sec9-t-N701-15May14_S1_Aligned.sorted.bam -b CA12k_Nov2014_V7_hg19.bed > coverage.BA8-t.V7.txt & /home/mzcai/softwares/bedtools-2.20.1/bin/coverageBed -counts -abam /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR/RL-BA8-sec9-n-N702-15May14_S2_mapped/RL-BA8-sec9-n-N702-15May14_S2_Aligned.sorted.bam -b CA12k_Nov2014_V7_hg19.bed > coverage.BA8-n.V7.txt &
- Sum up counts for each gene: CollapseSum_BedtoolsCoverageCounts.pl
- e.g. coverage.BA8-n.V7.txt > coverage.BA8-n.V7.collapsed.txt