Matt:LabNotes/2014-7-30

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Revision as of 07:09, 1 August 2014 by >Mzcai
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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";

Download resources from broadinstitute

  • ~/Genomes/RNA-SeQCResources
  • Download human rRNA reference file
 human_all_rRNA.fasta
  • Download GENCODE GTF file
 gencode.v7.annotation.gtf
  • Download GENCODE GC definitions file
 gencode.v7.gc.txt
 java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/ -BWArRNA ~/Genomes/RNA-SeqCResources/human_all_rRNA.fasta -s ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -t ~/Genomes/RNA-SeqCResources/gencode.v7.annotation.gtf -r ~/Genomes/HsGenome19.fa -o ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/ -strat gc -gc ~/Genomes/RNA-SeqCResources/gencode.v7.gc.txt -singleEnd
 
 org.broadinstitute.sting.utils.exceptions.UserException$LexicographicallySortedSequenceDictionary: Lexicographically sorted human genome sequence detected in reads.

For safety's sake the GATK requires human contigs in karyotypic order: 1, 2, ..., 10, 11, ..., 20, 21, 22, X, Y with M either leading or trailing these contigs. This is because all distributed GATK resources are sorted in karyotypic order, and your processing will fail when you need to use these files. You can use the ReorderSam utility to fix this problem: http://www.broadinstitute.org/gsa/wiki/index.php/ReorderSam