Matt:LabNotes/2014-7-30: Difference between revisions
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==RNA-SeQC== | ==RNA-SeQC== | ||
*Tophat aligned to hg19 BAM input | |||
===Trial 1=== | |||
====Add Read Groups and '''coordinate sort'''==== | |||
*Picard.AddOrReplaceReadGroups | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_RanHex.coorsortRG.bam' SORT_ORDER=coordinate RGID='1' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='RanHex' | java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_RanHex.coorsortRG.bam' SORT_ORDER=coordinate RGID='1' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='RanHex' | ||
====Index reference fasta and create sequence dictionary==== | |||
*reference fasta index already exists | |||
/home/kunzhang/HsGenome/hg19/HsGenome19.fa.fai | /home/kunzhang/HsGenome/hg19/HsGenome19.fa.fai | ||
*Picard.CreateSequenceDictionary | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/CreateSequenceDictionary.jar REFERENCE=/home/kunzhang/HsGenome/hg19/HsGenome19.fa OUTPUT=/home/mzcai/Genomes/HsGenome19.dict | java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/CreateSequenceDictionary.jar REFERENCE=/home/kunzhang/HsGenome/hg19/HsGenome19.fa OUTPUT=/home/mzcai/Genomes/HsGenome19.dict | ||
====Match order of contigs in Bam file to reference file==== | |||
*Picard.ReorderSam | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_RanHex.coorsortRG.bam OUTPUT=accepted_hits_RanHex.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa | java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_RanHex.coorsortRG.bam OUTPUT=accepted_hits_RanHex.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa | ||
====Index bam file==== | |||
samtools index accepted_hits_RanHex.coorsortRGreorder.bam | samtools index accepted_hits_RanHex.coorsortRGreorder.bam | ||
====Create sample file==== | |||
*~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt | |||
Sample ID Bam File Notes | Sample ID Bam File Notes | ||
RanHex /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam Indx26RanHex | RanHex /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam Indx26RanHex | ||
====Run RNA-SeQC v1.1.7.jar==== | |||
java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/bwa -BWArRNA /home/mzcai/scratch/RanHex_EnrichmRNA/mart_export_exons_totalrRNA.fa -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output -r /home/mzcai/Genomes/HsGenome19.fa -s /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -singleEnd -t /home/mzcai/LTS/Genomes/Homo_sapiens.GRCh37.75.chr.gtf -ttype 2 | java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/bwa -BWArRNA /home/mzcai/scratch/RanHex_EnrichmRNA/mart_export_exons_totalrRNA.fa -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output -r /home/mzcai/Genomes/HsGenome19.fa -s /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -singleEnd -t /home/mzcai/LTS/Genomes/Homo_sapiens.GRCh37.75.chr.gtf -ttype 2 | ||
The required transcript_id attribute was not found on line chr1 pseudogene gene 11869 14412 . + . gene_id "ENSG00000223972"; gene_name "DDX11L1"; gene_source "ensembl_havana"; gene_biotype "pseudogene"; | The required transcript_id attribute was not found on line chr1 pseudogene gene 11869 14412 . + . gene_id "ENSG00000223972"; gene_name "DDX11L1"; gene_source "ensembl_havana"; gene_biotype "pseudogene"; | ||
===Download resources from [http://www.broadinstitute.org/cancer/cga/rnaseqc_download broadinstitute]=== | ===Trial 2=== | ||
*Use provided gencode gtf annotation file | |||
**Also use provided rRNA ref file and GC definitions file to be consistent with example | |||
====Download resources from [http://www.broadinstitute.org/cancer/cga/rnaseqc_download broadinstitute]==== | |||
*~/Genomes/RNA-SeQCResources | *~/Genomes/RNA-SeQCResources | ||
*Download human rRNA reference file | *Download human rRNA reference file | ||
Line 32: | Line 43: | ||
gencode.v7.gc.txt | gencode.v7.gc.txt | ||
====Run RNA-SeQC v1.1.7.jar==== | |||
java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/ -BWArRNA ~/Genomes/RNA-SeqCResources/human_all_rRNA.fasta -s ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -t ~/Genomes/RNA-SeqCResources/gencode.v7.annotation.gtf -r ~/Genomes/HsGenome19.fa -o ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/ -strat gc -gc ~/Genomes/RNA-SeqCResources/gencode.v7.gc.txt -singleEnd | java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/ -BWArRNA ~/Genomes/RNA-SeqCResources/human_all_rRNA.fasta -s ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -t ~/Genomes/RNA-SeqCResources/gencode.v7.annotation.gtf -r ~/Genomes/HsGenome19.fa -o ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/ -strat gc -gc ~/Genomes/RNA-SeqCResources/gencode.v7.gc.txt -singleEnd | ||
org.broadinstitute.sting.utils.exceptions.UserException$LexicographicallySortedSequenceDictionary: Lexicographically sorted human genome sequence detected in reads. | org.broadinstitute.sting.utils.exceptions.UserException$LexicographicallySortedSequenceDictionary: Lexicographically sorted human genome sequence detected in reads. | ||
For safety's sake the GATK requires human contigs in karyotypic order: 1, 2, ..., 10, 11, ..., 20, 21, 22, X, Y with M either leading or trailing these contigs. | For safety's sake the GATK requires human contigs in karyotypic order: 1, 2, ..., 10, 11, ..., 20, 21, 22, X, Y with M either leading or trailing these contigs. | ||
This is because all distributed GATK resources are sorted in karyotypic order, and your processing will fail when you need to use these files. | This is because all distributed GATK resources are sorted in karyotypic order, and your processing will fail when you need to use these files. | ||
You can use the ReorderSam utility to fix this problem: http://www.broadinstitute.org/gsa/wiki/index.php/ReorderSam | You can use the ReorderSam utility to fix this problem: http://www.broadinstitute.org/gsa/wiki/index.php/ReorderSam | ||
===Reorder HsGenome19.fa to karyotypic order=== | ===Trial 3=== | ||
====Reorder HsGenome19.fa to karyotypic order==== | |||
csplit -f c HsGenome19.fa /'>chr9'/ /'>chr5'/ /'>chr2'/ /'>chr17'/ /'>chr14'/ /'>chr4'/ /'>chr16'/ /'>chr21'/ /'>chrM'/ /'>chr7'/ /'>chr3'/ /'>chr18'/ /'>chr12'/ /'>chrX'/ /'>chr13'/ /'>chr15'/ /'>chr8'/ /'>chr22'/ /'>chr11'/ /'>chr10'/ /'>chr20'/ /'>chr19'/ /'>chr6'/ /'>chr1'/ /'>chrY'/ | csplit -f c HsGenome19.fa /'>chr9'/ /'>chr5'/ /'>chr2'/ /'>chr17'/ /'>chr14'/ /'>chr4'/ /'>chr16'/ /'>chr21'/ /'>chrM'/ /'>chr7'/ /'>chr3'/ /'>chr18'/ /'>chr12'/ /'>chrX'/ /'>chr13'/ /'>chr15'/ /'>chr8'/ /'>chr22'/ /'>chr11'/ /'>chr10'/ /'>chr20'/ /'>chr19'/ /'>chr6'/ /'>chr1'/ /'>chrY'/ | ||
cat c24 c03 c11 c06 c02 c23 c10 c17 c01 c20 c19 c13 c15 c05 c16 c07 c04 c12 c22 c21 c08 c18 c14 c25 c09 > HsGenome19_karyorder.fa | cat c24 c03 c11 c06 c02 c23 c10 c17 c01 c20 c19 c13 c15 c05 c16 c07 c04 c12 c22 c21 c08 c18 c14 c25 c09 > HsGenome19_karyorder.fa | ||
rm HsGenome19.* | |||
mv HsGenome19_karyorder.fa ./HsGenome19.fa | |||
{| {{table}} | {| {{table}} | ||
Line 101: | Line 115: | ||
| >chrY||/'>chrY'/||c25||c09||chrM | | >chrY||/'>chrY'/||c25||c09||chrM | ||
|} | |} | ||
====Index reference fasta and create sequence dictionary==== | |||
*samtools faidx | |||
samtools faidx ~/Genomes/HsGenome19.fa | |||
*Picard.CreateSequenceDictionary | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/CreateSequenceDictionary.jar REFERENCE=~/Genomes/HsGenome19.fa OUTPUT=~/Genomes/HsGenome19.dict | |||
====Match order of contigs in Bam file to reference file==== | |||
*Picard.ReorderSam | |||
rm accepted_hits_RanHex.coorsortRGreorder.bam* | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_RanHex.coorsortRG.bam OUTPUT=accepted_hits_RanHex.coorsortRGreorder.bam REFERENCE=~/Genomes/HsGenome19.fa | |||
====Index bam file==== | |||
samtools index accepted_hits_RanHex.coorsortRGreorder.bam | |||
====Run RNA-SeQC v1.1.7.jar==== | |||
java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/bwa -BWArRNA ~/Genomes/RNA-SeqCResources/human_all_rRNA.fasta -s ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -t ~/Genomes/RNA-SeqCResources/gencode.v7.annotation.gtf -r ~/Genomes/HsGenome19.fa -o ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/ -strat gc -gc ~/Genomes/RNA-SeqCResources/gencode.v7.gc.txt -singleEnd | |||
<!-- | |||
RNA-SeQC v1.1.7 05/14/12 | |||
Retriving contig names from reference | |||
contig names in reference: 25 | |||
Loading GTF for Read Counting | |||
Converting to refGene | |||
Transcript objects to RefGen format: 2 s | |||
Running IntronicExpressionReadBlock Walker .... | |||
Arguments: [-T, IntronicExpressionReadBlock, --outfile_metrics, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/RanHex.metrics.tmp.txt, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -refseq, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//refGene.txt, -l, ERROR] | |||
Finished writing /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/RanHex.metrics.tmp.txt.intronReport.txt | |||
Finished writing /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/RanHex.metrics.tmp.txt.intronReport.txt_intronOnly.txt, now creating RPKM values for introns .. | |||
GATK command result code: 0 | |||
... GATK CoutReadMetrics Analysis DONE | |||
CountReadMetricsWalker Runtime: 0 min | |||
Counting rRNA reads with BWA and /home/mzcai/Genomes/RNA-SeqCResources/human_all_rRNA.fasta | |||
BWA on end 1 | |||
Running BWA on /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam | |||
Command: [/home/kunzhang/softwares/bwa-0.7.5a/bwa, aln, /home/mzcai/Genomes/RNA-SeqCResources/human_all_rRNA.fasta, -b0, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam] | |||
[bwa_aln] 17bp reads: max_diff = 2 | |||
[bwa_aln] 38bp reads: max_diff = 3 | |||
[bwa_aln] 64bp reads: max_diff = 4 | |||
[bwa_aln] 93bp reads: max_diff = 5 | |||
[bwa_aln] 124bp reads: max_diff = 6 | |||
[bwa_aln] 157bp reads: max_diff = 7 | |||
[bwa_aln] 190bp reads: max_diff = 8 | |||
[bwa_aln] 225bp reads: max_diff = 9 | |||
[bwa_aln_core] calculate SA coordinate... Call to BWA complete | |||
Running BWA sampe | |||
Command: [/home/kunzhang/softwares/bwa-0.7.5a/bwa, samse, /home/mzcai/Genomes/RNA-SeqCResources/human_all_rRNA.fasta, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/end1.sai, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam] | |||
[fread] Unexpected end of file | |||
Call to BWA complete | |||
Counting aligned reads in /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/rRNA.sam | |||
BWA based rRNA Estimation for RanHex: 0 min | |||
Calculating library complexity for RanHex | |||
Libary Complexity Calculation Time: 0 s | |||
Stratifying Transcripts By Expression | |||
Number of expressed transcripts at this cuttoff: 3093 | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/lowexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/RanHex/lowexpr | |||
Loading transcripts | |||
Preparing intervals for 1000 transcripts | |||
Interval Loading: 19 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/medexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/RanHex/medexpr | |||
Loading transcripts | |||
Preparing intervals for 1000 transcripts | |||
Interval Loading: 20 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/highexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/RanHex/highexpr | |||
Loading transcripts | |||
Preparing intervals for 1000 transcripts | |||
Interval Loading: 19 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/lowexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/lowexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/lowexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/lowexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 1 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/medexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/medexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/medexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/medexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 0 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/highexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/highexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/highexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/highexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 0 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/lowexpr//perBaseDoC.out: 4 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/lowexpr//perBaseDoC.out: 13 s | |||
Mapped intervals back to transcripts: 17 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/RanHex/lowexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/RanHex/lowexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/medexpr//perBaseDoC.out: 1 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/medexpr//perBaseDoC.out: 6 s | |||
Mapped intervals back to transcripts: 7 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/RanHex/medexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/RanHex/medexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/highexpr//perBaseDoC.out: 0 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//RanHex/highexpr//perBaseDoC.out: 1 s | |||
Mapped intervals back to transcripts: 2 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/RanHex/highexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/RanHex/highexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Lower Bound Z score cutoff: 0.375 | |||
Percentile: 0.03766231 | |||
Total Transcripts in this percentile: 3179 | |||
Upper Bound Z score cutoff: 0.625 | |||
Percentile: 0.090299495 | |||
Total Transcripts in this percentile: 7622 | |||
Middle Percentile: 0.8720382 | |||
Total Transcripts in this percentile: 73607 | |||
Copying transcript model in to GC stratifications | |||
Stratifying transcripts with file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/lowgc.gtf | |||
Arguments in this stratification [-bwa, /home/kunzhang/softwares/bwa-0.7.5a/bwa, -BWArRNA, /home/mzcai/Genomes/RNA-SeqCResources/human_all_rRNA.fasta, -s, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt, -t, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/lowgc.gtf, -r, /home/mzcai/Genomes/HsGenome19.fa, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low, -strat, none, -gc, /home/mzcai/Genomes/RNA-SeqCResources/gencode.v7.gc.txt, -singleEnd, -noReadCounting, -rRNA, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//rRNA_intervals.list, -expr, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//exons.rpkm.gct] | |||
RNA-SeQC v1.1.7 05/14/12 | |||
Suppressing Read Count Metrics within Recursive Call. | |||
Retriving contig names from reference | |||
contig names in reference: 25 | |||
Loading GTF for Read Counting | |||
Converting to refGene | |||
Transcript objects to RefGen format: 0 s | |||
Filtering GTF file to correspond to GCT file. | |||
Metrics suppressed | |||
Stratifying Transcripts By Expression | |||
Number of expressed transcripts at this cuttoff: 103 | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/lowexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/low/RanHex/lowexpr | |||
Loading transcripts | |||
Preparing intervals for 103 transcripts | |||
Interval Loading: 0 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/medexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/low/RanHex/medexpr | |||
Loading transcripts | |||
Preparing intervals for 103 transcripts | |||
Interval Loading: 0 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/highexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/low/RanHex/highexpr | |||
Loading transcripts | |||
Preparing intervals for 103 transcripts | |||
Interval Loading: 0 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/lowexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/lowexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/lowexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/lowexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 0 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/medexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/medexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/medexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/medexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 0 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/highexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/highexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/highexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/highexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 0 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/lowexpr//perBaseDoC.out: 0 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/lowexpr//perBaseDoC.out: 0 s | |||
Mapped intervals back to transcripts: 1 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/low/RanHex/lowexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/low/RanHex/lowexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/medexpr//perBaseDoC.out: 0 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/medexpr//perBaseDoC.out: 0 s | |||
Mapped intervals back to transcripts: 1 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/low/RanHex/medexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/low/RanHex/medexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/highexpr//perBaseDoC.out: 0 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/low/RanHex/highexpr//perBaseDoC.out: 0 s | |||
Mapped intervals back to transcripts: 1 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/low/RanHex/highexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/low/RanHex/highexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Stratifying transcripts with file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/medgc.gtf | |||
Arguments in this stratification [-bwa, /home/kunzhang/softwares/bwa-0.7.5a/bwa, -BWArRNA, /home/mzcai/Genomes/RNA-SeqCResources/human_all_rRNA.fasta, -s, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt, -t, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/medgc.gtf, -r, /home/mzcai/Genomes/HsGenome19.fa, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid, -strat, none, -gc, /home/mzcai/Genomes/RNA-SeqCResources/gencode.v7.gc.txt, -singleEnd, -noReadCounting, -rRNA, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//rRNA_intervals.list, -expr, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//exons.rpkm.gct] | |||
RNA-SeQC v1.1.7 05/14/12 | |||
Suppressing Read Count Metrics within Recursive Call. | |||
Retriving contig names from reference | |||
contig names in reference: 25 | |||
Loading GTF for Read Counting | |||
Converting to refGene | |||
Transcript objects to RefGen format: 0 s | |||
Filtering GTF file to correspond to GCT file. | |||
Metrics suppressed | |||
Stratifying Transcripts By Expression | |||
Number of expressed transcripts at this cuttoff: 2327 | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/lowexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/mid/RanHex/lowexpr | |||
Loading transcripts | |||
Preparing intervals for 1000 transcripts | |||
Interval Loading: 13 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/medexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/mid/RanHex/medexpr | |||
Loading transcripts | |||
Preparing intervals for 1000 transcripts | |||
Interval Loading: 13 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/highexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/mid/RanHex/highexpr | |||
Loading transcripts | |||
Preparing intervals for 1000 transcripts | |||
Interval Loading: 13 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/lowexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/lowexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/lowexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/lowexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 1 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/medexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/medexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/medexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/medexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 0 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/highexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/highexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/highexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/highexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 0 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/lowexpr//perBaseDoC.out: 2 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/lowexpr//perBaseDoC.out: 13 s | |||
Mapped intervals back to transcripts: 16 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/mid/RanHex/lowexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/mid/RanHex/lowexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/medexpr//perBaseDoC.out: 1 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/medexpr//perBaseDoC.out: 7 s | |||
Mapped intervals back to transcripts: 8 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/mid/RanHex/medexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/mid/RanHex/medexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/highexpr//perBaseDoC.out: 0 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/mid/RanHex/highexpr//perBaseDoC.out: 2 s | |||
Mapped intervals back to transcripts: 3 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/mid/RanHex/highexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/mid/RanHex/highexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Stratifying transcripts with file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/highgc.gtf | |||
Arguments in this stratification [-bwa, /home/kunzhang/softwares/bwa-0.7.5a/bwa, -BWArRNA, /home/mzcai/Genomes/RNA-SeqCResources/human_all_rRNA.fasta, -s, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt, -t, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/highgc.gtf, -r, /home/mzcai/Genomes/HsGenome19.fa, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high, -strat, none, -gc, /home/mzcai/Genomes/RNA-SeqCResources/gencode.v7.gc.txt, -singleEnd, -noReadCounting, -rRNA, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//rRNA_intervals.list, -expr, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//exons.rpkm.gct] | |||
RNA-SeQC v1.1.7 05/14/12 | |||
Suppressing Read Count Metrics within Recursive Call. | |||
Retriving contig names from reference | |||
contig names in reference: 25 | |||
Loading GTF for Read Counting | |||
Converting to refGene | |||
Transcript objects to RefGen format: 0 s | |||
Filtering GTF file to correspond to GCT file. | |||
Metrics suppressed | |||
Stratifying Transcripts By Expression | |||
Number of expressed transcripts at this cuttoff: 35 | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/lowexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/high/RanHex/lowexpr | |||
Loading transcripts | |||
Preparing intervals for 35 transcripts | |||
Interval Loading: 1 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/medexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/high/RanHex/medexpr | |||
Loading transcripts | |||
Preparing intervals for 35 transcripts | |||
Interval Loading: 1 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Expression file for DoC: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/highexpr/RanHex.transcripts.list | |||
Writing DoC per gene into: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/high/RanHex/highexpr | |||
Loading transcripts | |||
Preparing intervals for 35 transcripts | |||
Interval Loading: 1 s | |||
Creating interval list | |||
Writing intervals from transcript objects | |||
Transcript objects to interval list conversion: 0 s | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/lowexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/lowexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/lowexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/lowexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 0 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/medexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/medexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/medexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/medexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 0 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Running GATK Depth of Coverage Analysis .... | |||
Arguments: -T DepthOfCoverage -R /home/mzcai/Genomes/HsGenome19.fa -I /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/highexpr//perBaseDoC.out -L /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/highexpr/intervals.list -l ERROR | |||
Arguments Array: [-T, DepthOfCoverage, -R, /home/mzcai/Genomes/HsGenome19.fa, -I, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam, -o, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/highexpr//perBaseDoC.out, -L, /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/highexpr/intervals.list, -l, ERROR] | |||
GATK command result code: 0 | |||
Depth of Coverage run time: 0 min | |||
... GATK Depth of Coverage Analysis DONE | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/lowexpr//perBaseDoC.out: 0 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/lowexpr//perBaseDoC.out: 0 s | |||
Mapped intervals back to transcripts: 0 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/high/RanHex/lowexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/high/RanHex/lowexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/medexpr//perBaseDoC.out: 0 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/medexpr//perBaseDoC.out: 0 s | |||
Mapped intervals back to transcripts: 0 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/high/RanHex/medexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/high/RanHex/medexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Loading transcripts | |||
Splitting intervals into transcript-oriented DoC files | |||
Indexing DoC result file: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/highexpr//perBaseDoC.out: 0 s | |||
Finding DoC results per transcript | |||
DoC Results by interval have been mapped back to the transcripts: /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output//gc/high/RanHex/highexpr//perBaseDoC.out: 0 s | |||
Mapped intervals back to transcripts: 0 s | |||
Library size link path/home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/high/RanHex/highexpr/.. | |||
Absolute path to metrics.tmp.txt/media/Ext12T/MC_Ext12T/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/../home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/gc/high/RanHex/highexpr/RanHex.metrics.tmp.txt.rpkm.gct | |||
Finished Successfully. | |||
RNA-SeQC Total Runtime: 12 min--> | |||
===Trial 4: Add Other Samples=== | |||
*In respective directories | |||
**eg. ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx27_dT/ | |||
====Add Read Groups and '''coordinate sort'''==== | |||
*Picard.AddOrReplaceReadGroups | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_dT.coorsortRG.bam' SORT_ORDER=coordinate RGID='2' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='dT' | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_FISSEQRT.coorsortRG.bam' SORT_ORDER=coordinate RGID='3' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='FISSEQRT' | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_Top48.coorsortRG.bam' SORT_ORDER=coordinate RGID='4' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='Top48' | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_gDNA.coorsortRG.bam' SORT_ORDER=coordinate RGID='5' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='gDNA' | |||
====Match order of contigs in Bam file to reference file==== | |||
*Picard.ReorderSam | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_dT.coorsortRG.bam OUTPUT=accepted_hits_dT.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_FISSEQRT.coorsortRG.bam OUTPUT=accepted_hits_FISSEQRT.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_Top48.coorsortRG.bam OUTPUT=accepted_hits_Top48.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa | |||
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_gDNA.coorsortRG.bam OUTPUT=accepted_hits_gDNA.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa | |||
====Index bam file==== | |||
samtools index accepted_hits_dT.coorsortRGreorder.bam | |||
samtools index accepted_hits_FISSEQRT.coorsortRGreorder.bam | |||
samtools index accepted_hits_Top48.coorsortRGreorder.bam | |||
samtools index accepted_hits_gDNA.coorsortRGreorder.bam | |||
====Create sample file==== | |||
*~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt | |||
Sample ID Bam File Notes | |||
RanHex /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam Indx26RanHex | |||
dT /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx27_dT/accepted_hits_dT.coorsortRGreorder.bam Indx27dT | |||
FISSEQRT /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx28_FISSEQRT/accepted_hits_FISSEQRT.coorsortRGreorder.bam Indx28FISSEQRT | |||
Top48 /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx29_Top48/accepted_hits_Top48.coorsortRGreorder.bam Indx29Top48 | |||
gDNA /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx32_gDNA/accepted_hits_gDNA.coorsortRGreorder.bam Indx32gDNA | |||
====Run RNA-SeQC v1.1.7.jar==== | |||
java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/bwa -BWArRNA ~/Genomes/RNA-SeqCResources/human_all_rRNA.fasta -s ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -t ~/Genomes/RNA-SeqCResources/gencode.v7.annotation.gtf -r ~/Genomes/HsGenome19.fa -o ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_outputALL/ -strat gc -gc ~/Genomes/RNA-SeqCResources/gencode.v7.gc.txt -singleEnd | |||
*rRNA and rRNA rate values for all samples in index.html are "NA" |
Latest revision as of 22:32, 1 August 2014
RNA-SeQC[edit]
- Tophat aligned to hg19 BAM input
Trial 1[edit]
Add Read Groups and coordinate sort[edit]
- Picard.AddOrReplaceReadGroups
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_RanHex.coorsortRG.bam' SORT_ORDER=coordinate RGID='1' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='RanHex'
Index reference fasta and create sequence dictionary[edit]
- reference fasta index already exists
/home/kunzhang/HsGenome/hg19/HsGenome19.fa.fai
- Picard.CreateSequenceDictionary
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/CreateSequenceDictionary.jar REFERENCE=/home/kunzhang/HsGenome/hg19/HsGenome19.fa OUTPUT=/home/mzcai/Genomes/HsGenome19.dict
Match order of contigs in Bam file to reference file[edit]
- Picard.ReorderSam
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_RanHex.coorsortRG.bam OUTPUT=accepted_hits_RanHex.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa
Index bam file[edit]
samtools index accepted_hits_RanHex.coorsortRGreorder.bam
Create sample file[edit]
- ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt
Sample ID Bam File Notes RanHex /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam Indx26RanHex
Run RNA-SeQC v1.1.7.jar[edit]
java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/bwa -BWArRNA /home/mzcai/scratch/RanHex_EnrichmRNA/mart_export_exons_totalrRNA.fa -o /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output -r /home/mzcai/Genomes/HsGenome19.fa -s /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -singleEnd -t /home/mzcai/LTS/Genomes/Homo_sapiens.GRCh37.75.chr.gtf -ttype 2
The required transcript_id attribute was not found on line chr1 pseudogene gene 11869 14412 . + . gene_id "ENSG00000223972"; gene_name "DDX11L1"; gene_source "ensembl_havana"; gene_biotype "pseudogene";
Trial 2[edit]
- Use provided gencode gtf annotation file
- Also use provided rRNA ref file and GC definitions file to be consistent with example
Download resources from broadinstitute[edit]
- ~/Genomes/RNA-SeQCResources
- Download human rRNA reference file
human_all_rRNA.fasta
- Download GENCODE GTF file
gencode.v7.annotation.gtf
- Download GENCODE GC definitions file
gencode.v7.gc.txt
Run RNA-SeQC v1.1.7.jar[edit]
java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/ -BWArRNA ~/Genomes/RNA-SeqCResources/human_all_rRNA.fasta -s ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -t ~/Genomes/RNA-SeqCResources/gencode.v7.annotation.gtf -r ~/Genomes/HsGenome19.fa -o ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/ -strat gc -gc ~/Genomes/RNA-SeqCResources/gencode.v7.gc.txt -singleEnd org.broadinstitute.sting.utils.exceptions.UserException$LexicographicallySortedSequenceDictionary: Lexicographically sorted human genome sequence detected in reads. For safety's sake the GATK requires human contigs in karyotypic order: 1, 2, ..., 10, 11, ..., 20, 21, 22, X, Y with M either leading or trailing these contigs. This is because all distributed GATK resources are sorted in karyotypic order, and your processing will fail when you need to use these files. You can use the ReorderSam utility to fix this problem: http://www.broadinstitute.org/gsa/wiki/index.php/ReorderSam
Trial 3[edit]
Reorder HsGenome19.fa to karyotypic order[edit]
csplit -f c HsGenome19.fa /'>chr9'/ /'>chr5'/ /'>chr2'/ /'>chr17'/ /'>chr14'/ /'>chr4'/ /'>chr16'/ /'>chr21'/ /'>chrM'/ /'>chr7'/ /'>chr3'/ /'>chr18'/ /'>chr12'/ /'>chrX'/ /'>chr13'/ /'>chr15'/ /'>chr8'/ /'>chr22'/ /'>chr11'/ /'>chr10'/ /'>chr20'/ /'>chr19'/ /'>chr6'/ /'>chr1'/ /'>chrY'/ cat c24 c03 c11 c06 c02 c23 c10 c17 c01 c20 c19 c13 c15 c05 c16 c07 c04 c12 c22 c21 c08 c18 c14 c25 c09 > HsGenome19_karyorder.fa rm HsGenome19.* mv HsGenome19_karyorder.fa ./HsGenome19.fa
Old chr order | Add // | csplit output files | cat order | New chr order |
>chr9 | /'>chr9'/ | c01 | c24 | chr1 |
>chr5 | /'>chr5'/ | c02 | c03 | chr2 |
>chr2 | /'>chr2'/ | c03 | c11 | chr3 |
>chr17 | /'>chr17'/ | c04 | c06 | chr4 |
>chr14 | /'>chr14'/ | c05 | c02 | chr5 |
>chr4 | /'>chr4'/ | c06 | c23 | chr6 |
>chr16 | /'>chr16'/ | c07 | c10 | chr7 |
>chr21 | /'>chr21'/ | c08 | c17 | chr8 |
>chrM | /'>chrM'/ | c09 | c01 | chr9 |
>chr7 | /'>chr7'/ | c10 | c20 | chr10 |
>chr3 | /'>chr3'/ | c11 | c19 | chr11 |
>chr18 | /'>chr18'/ | c12 | c13 | chr12 |
>chr12 | /'>chr12'/ | c13 | c15 | chr13 |
>chrX | /'>chrX'/ | c14 | c05 | chr14 |
>chr13 | /'>chr13'/ | c15 | c16 | chr15 |
>chr15 | /'>chr15'/ | c16 | c07 | chr16 |
>chr8 | /'>chr8'/ | c17 | c04 | chr17 |
>chr22 | /'>chr22'/ | c18 | c12 | chr18 |
>chr11 | /'>chr11'/ | c19 | c22 | chr19 |
>chr10 | /'>chr10'/ | c20 | c21 | chr20 |
>chr20 | /'>chr20'/ | c21 | c08 | chr21 |
>chr19 | /'>chr19'/ | c22 | c18 | chr22 |
>chr6 | /'>chr6'/ | c23 | c14 | chrX |
>chr1 | /'>chr1'/ | c24 | c25 | chrY |
>chrY | /'>chrY'/ | c25 | c09 | chrM |
Index reference fasta and create sequence dictionary[edit]
- samtools faidx
samtools faidx ~/Genomes/HsGenome19.fa
- Picard.CreateSequenceDictionary
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/CreateSequenceDictionary.jar REFERENCE=~/Genomes/HsGenome19.fa OUTPUT=~/Genomes/HsGenome19.dict
Match order of contigs in Bam file to reference file[edit]
- Picard.ReorderSam
rm accepted_hits_RanHex.coorsortRGreorder.bam* java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_RanHex.coorsortRG.bam OUTPUT=accepted_hits_RanHex.coorsortRGreorder.bam REFERENCE=~/Genomes/HsGenome19.fa
Index bam file[edit]
samtools index accepted_hits_RanHex.coorsortRGreorder.bam
Run RNA-SeQC v1.1.7.jar[edit]
java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/bwa -BWArRNA ~/Genomes/RNA-SeqCResources/human_all_rRNA.fasta -s ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -t ~/Genomes/RNA-SeqCResources/gencode.v7.annotation.gtf -r ~/Genomes/HsGenome19.fa -o ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_output/ -strat gc -gc ~/Genomes/RNA-SeqCResources/gencode.v7.gc.txt -singleEnd
Trial 4: Add Other Samples[edit]
- In respective directories
- eg. ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx27_dT/
Add Read Groups and coordinate sort[edit]
- Picard.AddOrReplaceReadGroups
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_dT.coorsortRG.bam' SORT_ORDER=coordinate RGID='2' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='dT' java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_FISSEQRT.coorsortRG.bam' SORT_ORDER=coordinate RGID='3' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='FISSEQRT' java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_Top48.coorsortRG.bam' SORT_ORDER=coordinate RGID='4' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='Top48' java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/AddOrReplaceReadGroups.jar INPUT='accepted_hits.bam' OUTPUT='accepted_hits_gDNA.coorsortRG.bam' SORT_ORDER=coordinate RGID='5' RGLB='RTprimer' RGPL='ILLUMINA' RGPU='flowcell-barcode.lane' RGSM='gDNA'
Match order of contigs in Bam file to reference file[edit]
- Picard.ReorderSam
java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_dT.coorsortRG.bam OUTPUT=accepted_hits_dT.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_FISSEQRT.coorsortRG.bam OUTPUT=accepted_hits_FISSEQRT.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_Top48.coorsortRG.bam OUTPUT=accepted_hits_Top48.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa java -Xmx2g -jar /home/kunzhang/softwares/picard-tools-1.79/ReorderSam.jar INPUT=accepted_hits_gDNA.coorsortRG.bam OUTPUT=accepted_hits_gDNA.coorsortRGreorder.bam REFERENCE=/home/mzcai/Genomes/HsGenome19.fa
Index bam file[edit]
samtools index accepted_hits_dT.coorsortRGreorder.bam samtools index accepted_hits_FISSEQRT.coorsortRGreorder.bam samtools index accepted_hits_Top48.coorsortRGreorder.bam samtools index accepted_hits_gDNA.coorsortRGreorder.bam
Create sample file[edit]
- ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt
Sample ID Bam File Notes RanHex /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx26_RanHex/accepted_hits_RanHex.coorsortRGreorder.bam Indx26RanHex dT /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx27_dT/accepted_hits_dT.coorsortRGreorder.bam Indx27dT FISSEQRT /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx28_FISSEQRT/accepted_hits_FISSEQRT.coorsortRGreorder.bam Indx28FISSEQRT Top48 /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx29_Top48/accepted_hits_Top48.coorsortRGreorder.bam Indx29Top48 gDNA /home/mzcai/scratch/RanHex_EnrichmRNA/inVitroRTSeq/tophat_hg19unmask_Indx32_gDNA/accepted_hits_gDNA.coorsortRGreorder.bam Indx32gDNA
Run RNA-SeQC v1.1.7.jar[edit]
java -jar /home/kunzhang/softwares/RNA-SeQC_v1.1.7.jar -bwa /home/kunzhang/softwares/bwa-0.7.5a/bwa -BWArRNA ~/Genomes/RNA-SeqCResources/human_all_rRNA.fasta -s ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_samples.txt -t ~/Genomes/RNA-SeqCResources/gencode.v7.annotation.gtf -r ~/Genomes/HsGenome19.fa -o ~/scratch/RanHex_EnrichmRNA/inVitroRTSeq/RNA-SeQC_outputALL/ -strat gc -gc ~/Genomes/RNA-SeqCResources/gencode.v7.gc.txt -singleEnd
- rRNA and rRNA rate values for all samples in index.html are "NA"