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 | |||
==== | ====SortSam ver4==== | ||
*input file: bam | *input file: accepted_hits_##RG.rgroup.bam (output from previous step) | ||
*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: | *fasta file: Homo_sapiens_UCSC_hg19.fa | ||
*output prefix: | *output prefix: Homo_sapiens_UCSC_hg19 | ||
====Picard.CreateSequenceDictionary==== | ====Picard.CreateSequenceDictionary ver1==== | ||
*reference sequence file: | *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 | *input file: bam from SortSam | ||
*reference file: fa | *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