Over-Expressed Gene Sample

Question

[chimerajit]

I have few RNASeq samples where samples are carrying over expressed gene for e.g.

Sample1 = over expressed gene ABC, Sample2 = over expressed gene DBG ... etc.

and they also have a common control(which not carrying any over expressed gene)

Now do I just perform typical RNA-Seq analysis using Hisat ---->Stringtie-----> ballgown or I need to treat these samples special and do some standardization before ?

[chimerajit]

Thanks for your response. I also have a doubt and thus I asked. Thanks a lot.

[caggtaagtat]

Your welcome, I think, the effect on the rest of the genes will be very low though, if you don't include the expression vector in the reference genome during alignment.

Answer 1

[caggtaagtat]

Hi,

if this overexpression was induced by transfecting or transduction of cells with an expression vector, you should add the sequence of the resulting transcript in your reference genome during alignment of the reads. The reason for this is that sometimes, expression vectors use different UTRs than those which are naturally around the respective gene. This leads to reads which map partly to your gene of interest and partly to the "new" UTRs which can result in discarding of the respective reads and therefore an under-estimation of the actual expression of the gene of interest.