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