Matt:LabNotes/2014-8-1

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GenePattern: Tophat & RNA-SeQC[edit]

  • Using genepattern.broadinstitute.org

Tophat version 8.6[edit]

  • 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

Alignment Summary[edit]

  • RanHex_Indx26
 Reads:
         Input     :      5441
          Mapped   :      3765 (69.2% of input)
           of these:       384 (10.2%) have multiple alignments (18 have >20)
 69.2% overall read mapping rate.
  • dT_Indx27
 Reads:
         Input     :     10905
          Mapped   :      8444 (77.4% of input)
           of these:       838 ( 9.9%) have multiple alignments (65 have >20)
 77.4% overall read mapping rate.
  • FISSEQRT_Indx28
 Reads:
         Input     :      7024
          Mapped   :      5018 (71.4% of input)
           of these:       629 (12.5%) have multiple alignments (58 have >20)
 71.4% overall read mapping rate.
  • Top48_Indx29
 Reads:
         Input     :     12158
          Mapped   :      7220 (59.4% of input)
           of these:       801 (11.1%) have multiple alignments (72 have >20)
 59.4% overall read mapping rate.
  • gDNA_Indx32
 Reads:
         Input     :    111950
          Mapped   :     55280 (49.4% of input)
           of these:      6418 (11.6%) have multiple alignments (1749 have >20)
 49.4% overall read mapping rate.

RNA-SeQC[edit]

Picard.AddOrReplaceReadGroups ver3[edit]

  • input file: accepted_hits.bam
  • read group id: Indx##
  • read group library: RTenrichmRNA
  • read group platform: Illumina
  • read group platform unit: ## (eg 26 for Indx26)
  • read group sample name: (eg RanHex_Indx26)
  • output prefix: accepted_hits_##RG

SortSam ver4[edit]

  • input file: accepted_hits_##RG.rgroup.bam (output from previous step)
  • sort order: coordinate
  • output format: BAM
  • output prefix: accepted_hits_##

SAMtools.FastaIndex ver1.2[edit]

  • fasta file: Homo_sapiens_UCSC_hg19.fa
  • output prefix: Homo_sapiens_UCSC_hg19

Picard.CreateSequenceDictionary ver1[edit]

  • reference sequence file: Homo_sapiens_UCSC_hg19.fa
  • truncate names at white space: yes
  • output file: Homo_sapiens_UCSC_hg19.dict

Picard.ReorderSam[edit]

  • input file: bam from SortSam
  • reference file: fa from SAMtools.FastaIndex
  • reference sequence dictionary: Homo_sapiens_UCSC_hg19.dict from Picard.CreateSequenceDictionary

allow partial overlap: no allow contig length discordance: no output prefix: accepted_hits_##

Picard.MarkDuplicates[edit]

  • input file: reorder.bam from Picard.ReorderSam
  • remove duplicates: no
  • default everything
  • output prefix: accepted_hits_##

SortSam[edit]

  • Update index
  • input file: .mdup.bam from Picard.MarkDuplicate
  • sort order: coordinate
  • output format: BAM
  • output prefix: accepted_hits_##

RNAseqMetrics[edit]

  • bam files: zip of sorted.bam and sorted.bai files from SortSam
  • sample info file: <blank>
  • single end: yes
  • annotation gtf: Hg 19, GenCode,'chr1' format contigs
  • reference sequence: from SAMtools.FastaIndex
  • reference sequence index: from SAMtools.FastaIndex
  • reference sequence dictionary: from Picard.CreateSequenceDictionary
  • num genes: 1000
  • transcript level metrics: no
  • rRNA interval file: <empty>
  • rRNA reference file index: zip from BWA.indexer of human_all_rRNA.fa
 ~/Genomes/RNA-SeqCResources/human_all_rRNA.fasta input to BWA.indexer ver1.8 in GenePattern (is algorithm)
  • gc content file: <empty>
  • output prefix: RTenrichmRNA