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RNA-seq workshopALIGNMENT
Erin Osborne Nishimura
httponishwebuncHTSF-workshop-2015
Alignment with Tophat2
There are many alignment tools available
Nuno A Fonseca et al Bioinformatics 2012283169-3177copy The Author 2012 Published by Oxford University Press
Which aligner is best
bull What type of data do you have
bull What is your research question
Methods of alignment
bull Splice awarenessndash Splice unaware (Bowtie BWA)
ndash Faster
ndash Splice aware (Tophat MapSplice SpliceMap)ndash Slowerndash Yields more information on splice junctions
bull What will be matched firstndash Whole genomendash Known transcriptomendash A short segment of each read first
Why tophat
bull Popularbull Splice awarebull de novo or sequenced genome modesbull Transcriptome or whole genome assembly bull Lots of options for customization
bull Drawbacksndash Lots of parameters to set amp optimize
Tophat2 ndash how does it work
Kim et al 2013 Genome Biologyhttpwwwgenomebiologycom2013144r36
Tophat2 versus Tophat1
The good newshellip
bull hellip choice of aligner does not have a major impact on genes identified as differentially expressed compared to other choices
bull Fonseca 2014
Switch to Tophat2 Tutorial
bull httpsgithubcomerinosbHTSF_workshopblobmaster01_RNAseq_alignmentmd
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Alignment with Tophat2
There are many alignment tools available
Nuno A Fonseca et al Bioinformatics 2012283169-3177copy The Author 2012 Published by Oxford University Press
Which aligner is best
bull What type of data do you have
bull What is your research question
Methods of alignment
bull Splice awarenessndash Splice unaware (Bowtie BWA)
ndash Faster
ndash Splice aware (Tophat MapSplice SpliceMap)ndash Slowerndash Yields more information on splice junctions
bull What will be matched firstndash Whole genomendash Known transcriptomendash A short segment of each read first
Why tophat
bull Popularbull Splice awarebull de novo or sequenced genome modesbull Transcriptome or whole genome assembly bull Lots of options for customization
bull Drawbacksndash Lots of parameters to set amp optimize
Tophat2 ndash how does it work
Kim et al 2013 Genome Biologyhttpwwwgenomebiologycom2013144r36
Tophat2 versus Tophat1
The good newshellip
bull hellip choice of aligner does not have a major impact on genes identified as differentially expressed compared to other choices
bull Fonseca 2014
Switch to Tophat2 Tutorial
bull httpsgithubcomerinosbHTSF_workshopblobmaster01_RNAseq_alignmentmd
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
There are many alignment tools available
Nuno A Fonseca et al Bioinformatics 2012283169-3177copy The Author 2012 Published by Oxford University Press
Which aligner is best
bull What type of data do you have
bull What is your research question
Methods of alignment
bull Splice awarenessndash Splice unaware (Bowtie BWA)
ndash Faster
ndash Splice aware (Tophat MapSplice SpliceMap)ndash Slowerndash Yields more information on splice junctions
bull What will be matched firstndash Whole genomendash Known transcriptomendash A short segment of each read first
Why tophat
bull Popularbull Splice awarebull de novo or sequenced genome modesbull Transcriptome or whole genome assembly bull Lots of options for customization
bull Drawbacksndash Lots of parameters to set amp optimize
Tophat2 ndash how does it work
Kim et al 2013 Genome Biologyhttpwwwgenomebiologycom2013144r36
Tophat2 versus Tophat1
The good newshellip
bull hellip choice of aligner does not have a major impact on genes identified as differentially expressed compared to other choices
bull Fonseca 2014
Switch to Tophat2 Tutorial
bull httpsgithubcomerinosbHTSF_workshopblobmaster01_RNAseq_alignmentmd
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Which aligner is best
bull What type of data do you have
bull What is your research question
Methods of alignment
bull Splice awarenessndash Splice unaware (Bowtie BWA)
ndash Faster
ndash Splice aware (Tophat MapSplice SpliceMap)ndash Slowerndash Yields more information on splice junctions
bull What will be matched firstndash Whole genomendash Known transcriptomendash A short segment of each read first
Why tophat
bull Popularbull Splice awarebull de novo or sequenced genome modesbull Transcriptome or whole genome assembly bull Lots of options for customization
bull Drawbacksndash Lots of parameters to set amp optimize
Tophat2 ndash how does it work
Kim et al 2013 Genome Biologyhttpwwwgenomebiologycom2013144r36
Tophat2 versus Tophat1
The good newshellip
bull hellip choice of aligner does not have a major impact on genes identified as differentially expressed compared to other choices
bull Fonseca 2014
Switch to Tophat2 Tutorial
bull httpsgithubcomerinosbHTSF_workshopblobmaster01_RNAseq_alignmentmd
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Methods of alignment
bull Splice awarenessndash Splice unaware (Bowtie BWA)
ndash Faster
ndash Splice aware (Tophat MapSplice SpliceMap)ndash Slowerndash Yields more information on splice junctions
bull What will be matched firstndash Whole genomendash Known transcriptomendash A short segment of each read first
Why tophat
bull Popularbull Splice awarebull de novo or sequenced genome modesbull Transcriptome or whole genome assembly bull Lots of options for customization
bull Drawbacksndash Lots of parameters to set amp optimize
Tophat2 ndash how does it work
Kim et al 2013 Genome Biologyhttpwwwgenomebiologycom2013144r36
Tophat2 versus Tophat1
The good newshellip
bull hellip choice of aligner does not have a major impact on genes identified as differentially expressed compared to other choices
bull Fonseca 2014
Switch to Tophat2 Tutorial
bull httpsgithubcomerinosbHTSF_workshopblobmaster01_RNAseq_alignmentmd
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Why tophat
bull Popularbull Splice awarebull de novo or sequenced genome modesbull Transcriptome or whole genome assembly bull Lots of options for customization
bull Drawbacksndash Lots of parameters to set amp optimize
Tophat2 ndash how does it work
Kim et al 2013 Genome Biologyhttpwwwgenomebiologycom2013144r36
Tophat2 versus Tophat1
The good newshellip
bull hellip choice of aligner does not have a major impact on genes identified as differentially expressed compared to other choices
bull Fonseca 2014
Switch to Tophat2 Tutorial
bull httpsgithubcomerinosbHTSF_workshopblobmaster01_RNAseq_alignmentmd
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Tophat2 ndash how does it work
Kim et al 2013 Genome Biologyhttpwwwgenomebiologycom2013144r36
Tophat2 versus Tophat1
The good newshellip
bull hellip choice of aligner does not have a major impact on genes identified as differentially expressed compared to other choices
bull Fonseca 2014
Switch to Tophat2 Tutorial
bull httpsgithubcomerinosbHTSF_workshopblobmaster01_RNAseq_alignmentmd
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Tophat2 versus Tophat1
The good newshellip
bull hellip choice of aligner does not have a major impact on genes identified as differentially expressed compared to other choices
bull Fonseca 2014
Switch to Tophat2 Tutorial
bull httpsgithubcomerinosbHTSF_workshopblobmaster01_RNAseq_alignmentmd
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
The good newshellip
bull hellip choice of aligner does not have a major impact on genes identified as differentially expressed compared to other choices
bull Fonseca 2014
Switch to Tophat2 Tutorial
bull httpsgithubcomerinosbHTSF_workshopblobmaster01_RNAseq_alignmentmd
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Switch to Tophat2 Tutorial
bull httpsgithubcomerinosbHTSF_workshopblobmaster01_RNAseq_alignmentmd
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Generating genome browser tracks
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Ah those beautiful browser trackshellip
Brooks and Yang et al 2011 Genome Research
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
_trimfastq file
bamsam file
bw file
countstxt file
TOPHAT2
bedGraphToBigWig
bedtools genomecov
bg file
HTseq
DESeq2R
Differentially AbundantgenesIGVUCSC
Pretty browser shots
Todayrsquos simple analysis pipelinefastq file
trimmomaticbbduksh
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
I have included an example script for
bull Script05_makeBrowserTrackssh ndash Requires bedGraphToBigWigndash Requires bedtools
ndash Performs Normalizationbull Normalize to read depthbull One option
raquo Scale = (bps in genome)
(bp per read) x ( mapped reads)
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Two most common platforms
bull IGVndash httpswwwbroadinstituteorgigvndash Locally installed
bull UCSC Genome Browserndash httpgenomeucscedundash Upload required
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
Visual inspection of each normalized replicate is criticalhellip
bull httpgenomeucsceducgi-binhgTrackshgS_doOtherUser=submitamphgS_otherUserName=Erin20OsborneamphgS_otherUserSessionName=hg19_female_v_male_demo
bull httpbitly1SJrQcq
