Matt:LabNotes/2014-8-1: Difference between revisions

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**all others not mentioned were left at default
**all others not mentioned were left at default


====Alignment Summary====
*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===
===RNA-SeQC===
 
<!--====Picard.MergeBamAlignment====
====Picard.MergeBamAlignment====
*to add unaligned reads bam file to accepted
*to add unaligned reads bam file to accepted
-->
====Picard.AddOrReplaceReadGroups ver3====
*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


====Picard.AddOrReplaceReadGroups====
====SortSam ver4====
*input file: bam
*input file: accepted_hits_##RG.rgroup.bam (output from previous step)
*read group id:
 
====SortSam====
*input file:
*sort order: coordinate
*sort order: coordinate
output format: BAM
*output format: BAM
*output prefix:
*output prefix: accepted_hits_##


====SAMtools.FastaIndex====
====SAMtools.FastaIndex ver1.2====
*fasta file: reference.fa
*fasta file: Homo_sapiens_UCSC_hg19.fa
*output prefix:
*output prefix: Homo_sapiens_UCSC_hg19


====Picard.CreateSequenceDictionary====
====Picard.CreateSequenceDictionary ver1====
*reference sequence file: reference.fa
*reference sequence file: Homo_sapiens_UCSC_hg19.fa
*truncate names at white space: yes
*output file: Homo_sapiens_UCSC_hg19.dict


====Picard.ReorderSam====
====Picard.ReorderSam====
*input file: bam/bai pair from SortSam
*input file: bam from SortSam
*reference file: fa/fai from SAMtools.FastaIndex
*reference file: fa from SAMtools.FastaIndex
*reference sequence dictionary: .dict from Picard.CreateSequenceDictionary
*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====
====Picard.MarkDuplicates====
*input file: bam from Picard.ReorderSam
*input file: reorder.bam from Picard.ReorderSam
*remove duplicates: no
*remove duplicates: no
*default everything
*output prefix: accepted_hits_##


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


====RNAseqMetrics====
====RNAseqMetrics====
*bam files: from SortSam
*bam files: zip of sorted.bam and sorted.bai files from SortSam
*sample info file:
*sample info file: <blank>
*single end:
*single end: yes
*annotation gtf: Hg 19, GenCode
*annotation gtf: Hg 19, GenCode,'chr1' format contigs
*reference sequence: from SAMtools.FastaIndex
*reference sequence: from SAMtools.FastaIndex
*reference sequence index: from SAMtools.FastaIndex
*reference sequence index: from SAMtools.FastaIndex
Line 76: Line 119:
*num genes: 1000
*num genes: 1000
*transcript level metrics: no
*transcript level metrics: no
*gc content file:
*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

Latest revision as of 20:52, 8 August 2014

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