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Old 07-07-2014, 12:27 AM   #1
Parharn
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Default Question: How to merge replicates of mapped reads out of Tophat for pathview and gage

Hi, I have three biological replicates for my experiment. I wonder how should I merge the accepted_hits.bam out of Tophat for doing Pathview/gage analysis for "RNA-seq Pathway and Gene-set analysis workflow"? Let's name the accepted_hits.bam replicates as 1.bam 2.bam and 3.bam for the sake of short writing.

I have a thread on biostars.org
https://www.biostars.org/p/105503/
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Old 07-07-2014, 04:09 AM   #2
TiborNagy
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The documentation suggest you need to use a differential expression analysis solution: DESeq2, Cufflinks, Limma and after that use gage. These packages handle the replicates.
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Old 07-28-2014, 11:31 AM   #3
bigmw
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You would need to index all of them and get .bam.bai file for each one of them. Then collect all .bam and .bam.bai files into the same directory (i.e. tophat_all as in the demo code). Then all samples will be scanned in by function summarizeOverlaps at step 1: Count the reads mapped to each gene.
If you read carefully, actually all details are covered in Section 3.1 Preparation: read mapping and package installation:
http://www.bioconductor.org/packages...eqWorkflow.pdf


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Originally Posted by Parharn View Post
Hi, I have three biological replicates for my experiment. I wonder how should I merge the accepted_hits.bam out of Tophat for doing Pathview/gage analysis for "RNA-seq Pathway and Gene-set analysis workflow"? Let's name the accepted_hits.bam replicates as 1.bam 2.bam and 3.bam for the sake of short writing.

I have a thread on biostars.org
https://www.biostars.org/p/105503/
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Old 10-27-2014, 12:29 PM   #4
bigmw
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Thatís the joint workflow for the usersí convenience. But GAGE/Pathview does not really require a external differential expression analysis step, as shown in the native workflow. For details:
http://bioconductor.org/packages/rel...eqWorkflow.pdf


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Originally Posted by TiborNagy View Post
The documentation suggest you need to use a differential expression analysis solution: DESeq2, Cufflinks, Limma and after that use gage. These packages handle the replicates.
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Old 10-27-2014, 01:03 PM   #5
Parharn
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Yes, later I discovered it but thanks to saying anyway
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