Matt:LabNotes/2014-7-30

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Revision as of 00:57, 1 August 2014 by >Mzcai (Created page with "==RNA-SeQC== *Bam input **Add Read Groups and '''coordinate sort''' ***Picard.AddOrReplaceReadGroups java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplac...")
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RNA-SeQC

  • Bam input
    • Add Read Groups and coordinate sort
      • Picard.AddOrReplaceReadGroups
 java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_RanHex.coorsortRG.bam' SORT_ORDER=coordinate RGID='1' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='RanHex'
    • Index reference fasta and create sequence dictionary
      • reference fasta index already exists
 /home/kunzhang/HsGenome/hg19/HsGenome19.fa.fai
      • Picard.CreateSequenceDictionary
 java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/CreateSequenceDictionary.jar REFERENCE=/home/kunzhang/HsGenome/hg19/HsGenome19.fa OUTPUT=/home/mzcai/Genomes/HsGenome19.dict
    • Match order of contigs in Bam file to reference file
      • Picard.ReorderSam
 java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_RanHex.coorsortRG.bam OUTPUT=accepted_hits_RanHex.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa
    • Index bam file
 samtools index accepted_hits_RanHex.coorsortRGreorder.bam
  • Create sample file: ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt
 Sample ID       Bam File        Notes
 RanHex  /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam   Indx26RanHex
 java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/bwa -BWArRNA /home/mzcai/scratch/RanHex_EnrichmRNA/mart_export_exons_totalrRNA.fa -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output -r /home/mzcai/Genomes/HsGenome19.fa -s /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -singleEnd -t /home/mzcai/LTS/Genomes/Homo_sapiens.GRCh37.75.chr.gtf -ttype 2
 The required transcript_id attribute was not found on line chr1 pseudogene      gene    11869   14412   .       +       .       gene_id "ENSG00000223972"; gene_name "DDX11L1"; gene_source "ensembl_havana"; gene_biotype "pseudogene";