Getting Transcipt Variants For Specific Genes In Rna-Seq Data

Question

[Rubal7]

Hello Everyone,

I have detected a duplication in a population using whole-genome sequencing. Now I want to see if the duplicated version of the gene is also being expressed. Fortunately we have some expression data from the same individuals generated using RNA-SEQ. I have never handled RNA-SEQ data before but somebody else in the lab has been generating and processing this data. Is there a straightforward way (like a tool) to extract all the transcripts that have been mapped to a specific gene? The goal is to see if transcripts exist with the variants that we see in the exons of the duplicated version of the gene to know if it is being expressed.

Thanks in advance for your help!

Best, Rubal

Answer 1

[Chris Miller]

Assuming you've mapped your RNAseq data and have a bam file, you can use samtools mpileup to extract the base calls from a particular position. You could also just load your bam in IGV and view it manually.

[Ketil]

I don't think this will work without a bit of clever processing. If the gene is duplicated, a lot of reads will map equally well to both genes, and probably just be assigned to one of them randomly (depending on the aligner).

[Chris Miller]

If there are SNVs specific to the duplicated copy of the gene, then looking at the proportion of that snp at each site will tell you how frequently it was expressed, relative to the other alleles.

Answer 2

[JC]

Actually it could be a little complicated because the mapping will overlap all transcripts in one region. But it can be resolved if the duplication event produces different transcripts or has allele specific SNPs. For example, if your duplication deletes one or more exons, you will see the expression level of the whole gene is 2X but in the missing exons is only 1X, then you can use that evidence to compute the real expression level of each transcript. The allele SNP case is similar. To extract the reads mapped in a region and assuming you have the BAM file, you can use samtools directly:

samtols view file.bam chrA:start-end > region.bam

[Rubal7]

thanks some good ideas here, ill look for differential coverage between exons and for duplication specific snps in the RNA reads