Matt:LabNotes/2015-4-27: Difference between revisions

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>Mzcai
(Created page with "==Checking RNA-Seq Read Coverage of Probe Targets== *[[Matt:LabNotes/2014-11-1#Filter_Probes_with_Low_Coverage_based_on_Brain_RNA-seq_Data_from_Rui.2FBlue | Probes were alread...")
 
>Mzcai
mNo edit summary
 
(3 intermediate revisions by the same user not shown)
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*[[Media:CA12kNov2014_Probelist2BED.txt |CA12kNov2014_Probelist2BED.pl]]
*[[Media:CA12kNov2014_Probelist2BED.txt |CA12kNov2014_Probelist2BED.pl]]
**Made start and end positions +/- 25bp from target position
**Made start and end positions +/- 25bp from target position
**[[Media:OutputFile_0gap_contig_final.full_info.V4.txt|OutputFile_0gap_contig_final.full_info.V4.txt]] > [[Media::CA12k_Nov2014_V4.txt|CA12k_Nov2014_V4.bed]]
**[[Media:OutputFile_0gap_contig_final.full_info.V4.txt|OutputFile_0gap_contig_final.full_info.V4.txt]] > [[Media:CA12k_Nov2014_V4.txt|CA12k_Nov2014_V4.bed]]
**[[Media:OutputFile_0gap_final.full_info.V7.txt|OutputFile_0gap_final.full_info.V7.txt]] > CA12k_Nov2014_V7.bed
**[[Media:OutputFile_0gap_final.full_info.V7.txt|OutputFile_0gap_final.full_info.V7.txt]] > CA12k_Nov2014_V7.bed


*[[Media:ConvertProbesHg38_2_BedHg19.txt | ConvertProbesHg38_2_BedHg19.pl]]
*Convert from hg38 reference positions to hg19 reference (which RNA-Seq data was mapped to) using [http://genome.ucsc.edu/cgi-bin/hgLiftOver 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===
===Bedtools Coverage with RNA-Seq Data===
Line 18: Line 20:
*RNA-Seq data from: /media/LTS_33T/RL_LTS33T/201404_201405_7Samples_BulkNucleiBatch1-20140623_Expt146/STAR$
*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
**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
**RL-BA8-sec9-n-N702-15May14_S2_mapped/RL-BA8-sec9-n-N702-15May14_S2_Aligned.sorted.bam
***45,222,268 reads
*Commands
<!--
  /home/mzcai/softwares/bedtools-2.20.1/bin/coverageBed -hist -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.hist.BA8-t.V4.txt &
  /home/mzcai/softwares/bedtools-2.20.1/bin/coverageBed -hist -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.hist.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_V4_hg19.bed > coverage.counts.BA8-t.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-n-N702-15May14_S2_mapped/RL-BA8-sec9-n-N702-15May14_S2_Aligned.sorted.bam -b CA12k_Nov2014_V4_hg19.bed > coverage.counts.BA8-n.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-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 &


*Commands
*Sum up counts for each gene: CollapseSum_BedtoolsCoverageCounts.pl
  /home/mzcai/softwares/bedtools-2.20.1/bin/coverageBed -hist -abam RL-BA8-sec9-t-N701-15May14_S1_mapped/RL-BA8-sec9-t-N701-15May14_S1_Aligned.sorted.bam -b CA12k_Nov2014_V4.bed >
**e.g. coverage.BA8-n.V7.txt > coverage.BA8-n.V7.collapsed.txt
 
===Results===
[[Media:20150427_BulkSeqCounts_CA12kNov2014Targets.xlsx]]

Latest revision as of 19:41, 1 May 2015

Checking RNA-Seq Read Coverage of Probe Targets[edit]

Create BED File for Probe Targets[edit]

  • 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
  • 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

Results[edit]

Media:20150427_BulkSeqCounts_CA12kNov2014Targets.xlsx