Matt:LabNotes/2014-8-1

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Revision as of 16:38, 8 August 2014 by >Mzcai
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GenePattern: Tophat & RNA-SeQC

  • Using genepattern.broadinstitute.org

Tophat version 8.6

  • Upload files
 s_1_1_Indx26.txt
 s_1_1_Indx27.txt
 s_1_1_Indx28.txt
 s_1_1_Indx29.txt
 s_1_1_Indx32.txt
  • Bowtie index
 Homo_sapiens_hg19_UCSC
  • GTF file
 Homo_sapiens_GENCODE_hg19_v7_chr1_format.gtf
  • library type
 Standard Illumina (fr-unstranded)
  • quality value scale
 Solexa 1.3 (Phred 64)
  • output prefix
 <Indx##reads.pair.1_basename>
  • transcriptome only
 yes
  • left blank:
    • transcriptome index
    • reads pair 2
    • mate inner distance
    • mate std dev
    • all others not mentioned were left at default

RNA-SeQC

Picard.MergeBamAlignment

  • to add unaligned reads bam file to accepted

Picard.AddOrReplaceReadGroups

  • input file: bam
  • read group id:

SortSam

  • input file:
  • sort order: coordinate

output format: BAM

  • output prefix:

SAMtools.FastaIndex

  • fasta file: reference.fa
  • output prefix:

Picard.CreateSequenceDictionary

  • reference sequence file: reference.fa

Picard.ReorderSam

  • input file: bam/bai pair from SortSam
  • reference file: fa/fai from SAMtools.FastaIndex
  • reference sequence dictionary: .dict from Picard.CreateSequenceDictionary

Picard.MarkDuplicates

  • input file: bam from Picard.ReorderSam
  • remove duplicates: no

SortSam

  • Update index
  • input file: from Picard.MarkDuplicate
  • sort order: coordinate
  • output: BAM

RNAseqMetrics

  • bam files: from SortSam
  • sample info file:
  • single end:
  • annotation gtf: Hg 19, GenCode
  • reference sequence: from SAMtools.FastaIndex
  • reference sequence index: from SAMtools.FastaIndex
  • reference sequence dictionary: from Picard.CreateSequenceDictionary
  • num genes: 1000
  • transcript level metrics: no
  • gc content file: