Rna-Seq Mutation Detection

7.3 years ago by

Jeremy Goecks • 2.2k wrote:

Rich, Given that you're analyzing your RNA-seq data using Galaxy, I'd guess that you're using Tophat to map your reads onto on reference genome. If this is the case, then you can use the BAM files produced by Tophat to generate variation data for each sample. The variation tools that you'll want to look at are [NGS: SAM Tools-->]Generate Pileup [NGS: GATK Tools-->]Unified Genotyper (only avaiable on our test server and still in beta) The outputs for each tool produce a consensus base for each potential variation site, and you can compare the consensus base for each sample to look for differences. If you're doing de novo assembly of your RNA-seq data to look for variation, you'll need to use tools that are not currently available in Galaxy. Good luck, J.

ADD COMMENT • link written 7.3 years ago by Jeremy Goecks • 2.2k

Hi, Thanks very much. I've tried this, but one thing I have noticed is that if I do the initial mapping with BWA vs. Bowtie the # of variants I get is much larger with BWA. I have seen mention on the web that you need to change the quality score annotation for BWA before running SAMtools, but not sure precisely how to do this. any thoughts? Rich ________________________________ To: Richard Mark White <whiter3@yahoo.com> Cc: "galaxy-user@lists.bx.psu.edu" <galaxy-user@lists.bx.psu.edu> Subject: Re: [galaxy-user] rna-seq mutation detection Rich, Given that you're analyzing your RNA-seq data using Galaxy, I'd guess that you're using Tophat to map your reads onto on reference genome. If this is the case, then you can use the BAM files produced by Tophat to generate variation data for each sample. The variation tools that you'll want to look at are [NGS: SAM Tools-->]Generate Pileup [NGS: GATK Tools-->]Unified Genotyper (only avaiable on our test server and still in beta) The outputs for each tool produce a consensus base for each potential variation site, and you can compare the consensus base for each sample to look for differences. If you're doing de novo assembly of your RNA-seq data to look for variation, you'll need to use tools that are not currently available in Galaxy. Good luck, J. Hi,

ADD REPLY • link written 7.3 years ago by Richard Mark White • 240

Rich, You can convert base quality scores using the FASTQ Groomer tool. Note that Galaxy tools typically work with Sanger (Phred+33) quality scores. Good luck, J.

ADD REPLY • link written 7.3 years ago by Jeremy Goecks • 2.2k

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