Asboner: /* Alignment */

2010-07-08T05:23:18Z

Alignment

← Older revision		Revision as of 05:23, 8 July 2010
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	* Bowtie alignment of the RNA-Seq reads to the splice junction library		* Bowtie alignment of the RNA-Seq reads to the splice junction library
	bowtie -q -p 4 knownGene_2x20_spliceJunctions sample.fastq ~~sample_splices~~.bowtie		bowtie -q -p 4 knownGene_2x20_spliceJunctions sample.fastq sample_junctions.bowtie

	<br>		<br>

	<center>[[#top]]</center>		<center>[[#top]]</center>

	=== Conversion ===		=== Conversion ===

Asboner: /* Prerequisites */

2010-07-02T09:59:27Z

Prerequisites

← Older revision		Revision as of 09:59, 2 July 2010
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	* Build bowtie index for the reference genome		* Build bowtie index for the reference genome
	bowtie-build -f chr1.fa,chr10.fa,chr11.fa,chr12.fa,chr13.fa,chr14.fa,chr15.fa,chr16.fa,chr17.fa,chr18.fa,chr19.fa,		bowtie-build -f chr1.fa,chr10.fa,chr11.fa,chr12.fa,chr13.fa,chr14.fa,chr15.fa,chr16.fa,chr17.fa,chr18.fa,chr19.fa,
	chr20.fa,chr21.fa,chr22.fa,chr2,chr3.fa,chr4.fa,chr5.fa,chr6.fa,chr7.fa,chr8.fa,chr9.fa,chrX.fa,chrY.fa hg18		chr20.fa,chr21.fa,chr22.fa,chr2.fa,chr3.fa,chr4.fa,chr5.fa,chr6.fa,chr7.fa,chr8.fa,chr9.fa,chrX.fa,chrY.fa hg18

	* Build bowtie index for the splice junction library		* Build bowtie index for the splice junction library

Asboner: /* Prerequisites */

2010-06-30T08:44:08Z

Prerequisites

← Older revision		Revision as of 08:44, 30 June 2010
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	chr20.fa,chr21.fa,chr22.fa,chr2,chr3.fa,chr4.fa,chr5.fa,chr6.fa,chr7.fa,chr8.fa,chr9.fa,chrX.fa,chrY.fa hg18		chr20.fa,chr21.fa,chr22.fa,chr2,chr3.fa,chr4.fa,chr5.fa,chr6.fa,chr7.fa,chr8.fa,chr9.fa,chrX.fa,chrY.fa hg18

	* Build bowtie index for the splice junction ~~liabrary~~		* Build bowtie index for the splice junction library
	bowtie-build -f knownGene_2x20_spliceJunctions.fa knownGene_2x20_spliceJunctions		bowtie-build -f knownGene_2x20_spliceJunctions.fa knownGene_2x20_spliceJunctions

Infoadmin: Created page with '[http://archive.gersteinlab.org/proj/rnaseq/rseqtools '''RSEQtools Main Page'''] TOC == Example workflow == This section shows a simple workflow for proce…'

2010-06-09T11:57:51Z

Created page with '<center>[http://archive.gersteinlab.org/proj/rnaseq/rseqtools '''RSEQtools Main Page''']</center> __TOC__ == Example workflow == This section shows a simple workflow for proce…'

New page

<center>[http://archive.gersteinlab.org/proj/rnaseq/rseqtools '''RSEQtools Main Page''']</center>

__TOC__

== Example workflow ==

This section shows a simple workflow for processing a RNA-Seq data set. A sample data set that was originally published in [http://www.ncbi.nlm.nih.gov/pubmed/20194744 PNAS] is used for this example. Specifically, this sample data set (human embryonic stem cells) consists of 27 nucleotides single-end reads. The reads are aligned to the human reference genome (hg18) and to a splice junction library generated from the UCSC Known Genes annotation set using [http://bowtie-bio.sourceforge.net/index.shtml Bowtie].

 

<center>[[#top]]</center>
=== Prerequisites ===

* Software download:
** [https://sourceforge.net/projects/bowtie-bio/files/bowtie/0.12.5 Bowtie]

* Download the following data files:
** [http://hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/chromFa.zip chromFa.zip] - Human reference genome (hg18), one file per chromosome.
** [http://hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/hg18.2bit hg18.2bit] contains the complete hg18 Human Genome in the 2bit format.
** [http://hgdownload.cse.ucsc.edu/goldenPath/hg18/database/knownGene.txt.gz knownGene.txt.gz] - UCSC Known Genes annotation set.
** [http://hgdownload.cse.ucsc.edu/goldenPath/hg18/database/knownIsoforms.txt.gz knownIsoforms.txt.gz] Links together various transcripts (UCSC Known Genes) of a gene into a cluster.
** [http://archive.gersteinlab.org/proj/rnaseq/rseqtools/sample.fastq.gz sample.fastq.gz ] - Sample data (hESC; 27nt; single-end; 5 million reads, FASTQ format).

* Decompress the files
gunzip knownGene.txt.gz knownIsoforms.txt.gz
unzip chromFa.zip

* Convert knownGene.txt into the [[RSEQtools#Interval|Interval]] format
cut -f1,2,3,4,5,8,9,10 knownGene.txt > knownGene.interval

* Generate the composite gene models
[[RSEQtools#mergeTranscripts|mergeTranscripts]] knownIsoforms.txt knownGene.interval compositeModel > knownGene_composite.interval

* Create a splice junction library based on the UCSC annotation set
[[RSEQtools#createSpliceJunctionLibrary|createSpliceJunctionLibrary]] hg18.2bit knownGene.interval 20 > knownGene_2x20_spliceJunctions.fa

* Build bowtie index for the reference genome
bowtie-build -f chr1.fa,chr10.fa,chr11.fa,chr12.fa,chr13.fa,chr14.fa,chr15.fa,chr16.fa,chr17.fa,chr18.fa,chr19.fa,
chr20.fa,chr21.fa,chr22.fa,chr2,chr3.fa,chr4.fa,chr5.fa,chr6.fa,chr7.fa,chr8.fa,chr9.fa,chrX.fa,chrY.fa hg18

* Build bowtie index for the splice junction liabrary
bowtie-build -f knownGene_2x20_spliceJunctions.fa knownGene_2x20_spliceJunctions

 

<center>[[#top]]</center>

=== Alignment ===

* Bowtie alignment of the RNA-Seq reads to the human reference genome
bowtie -q -p 4 hg18 sample.fastq sample_hg18.bowtie

* Bowtie alignment of the RNA-Seq reads to the splice junction library
bowtie -q -p 4 knownGene_2x20_spliceJunctions sample.fastq sample_splices.bowtie

 

<center>[[#top]]</center>
=== Conversion ===

* Convert bowtie output to [[RSEQtools#MRF|MRF]]
[[RSEQtools#bowtie2mrf|bowtie2mrf]] genomic < sample_hg18.bowtie > sample_hg18.mrf
[[RSEQtools#bowtie2mrf|bowtie2mrf]] junctions < sample_junctions.bowtie > sample_junctions.mrf

 

<center>[[#top]]</center>
=== Downstream analyses ===

* Calculate gene expression values using the composite gene models
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfQuantifier|mrfQuantifier]] knownGene_composite.interval multipleOverlap > sample.geneExpression

* Generate a signal track files in [[RSEQtools#WIG|WIG]] format of the mapped reads
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrf2wig|mrf2wig]] sample

* Generate [[RSEQtools#GFF|GFF]] files representing the splice junctions
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrf2gff|mrf2gff]] sample

* Determine whether there is a mapping bias
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfMappingBias|mrfMappingBias]] knownGene_composite.interval > sample.mappingBias

* Calculate the annotation coverage for the UCSC KnownGene composite gene models
cat sample_hg18.mrf sample_junctions.mrf | wc -l
Result: 4760475

cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfAnnotationCoverage|mrfAnnotationCoverage]] knownGene_composite.interval 4760475 500000 1
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfAnnotationCoverage|mrfAnnotationCoverage]] knownGene_composite.interval 4760475 1000000 1
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfAnnotationCoverage|mrfAnnotationCoverage]] knownGene_composite.interval 4760475 1500000 1
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfAnnotationCoverage|mrfAnnotationCoverage]] knownGene_composite.interval 4760475 2000000 1
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfAnnotationCoverage|mrfAnnotationCoverage]] knownGene_composite.interval 4760475 2500000 1
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfAnnotationCoverage|mrfAnnotationCoverage]] knownGene_composite.interval 4760475 3000000 1
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfAnnotationCoverage|mrfAnnotationCoverage]] knownGene_composite.interval 4760475 3500000 1
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfAnnotationCoverage|mrfAnnotationCoverage]] knownGene_composite.interval 4760475 4000000 1
cat sample_hg18.mrf sample_junctions.mrf | [[RSEQtools#mrfAnnotationCoverage|mrfAnnotationCoverage]] knownGene_composite.interval 4760475 4500000 1

RSEQtools/workflow - Revision history

Asboner: /* Alignment */

Asboner: /* Prerequisites */

Asboner: /* Prerequisites */

Infoadmin: Created page with '[http://archive.gersteinlab.org/proj/rnaseq/rseqtools '''RSEQtools Main Page'''] __TOC__ == Example workflow == This section shows a simple workflow for proce…'

Infoadmin: Created page with '[http://archive.gersteinlab.org/proj/rnaseq/rseqtools '''RSEQtools Main Page'''] TOC == Example workflow == This section shows a simple workflow for proce…'