Split N Trim RNAseq data

[skbrimer]

Hello everyone,

I have more of a general pre-processing question. We are doing some small viral genome sequencing. The virus is ssRNA (coronaviridae) and we have been using the RNAseq method on the PGM and I was just wondering if I'm doing the pre-processing correctly.

I have been treating the reads like genomic DNA for the processing, which is just mapping to the reference and marking duplicates. Is this correct? Should I be looking for splits? I know that viruses have different splices to make early and late proteins, but not really these two genes make one protein splices.

The more I thought about it the more unsure I became, does anyone have an opinion?

[Brian Bushnell]

It seems prudent to map with a splice-aware aligner, just to be on the safe side.

[SylvainL]

Hi,

simply tell us with mapper you used. If you used bowtie2 it should be fine...

[skbrimer]

Hi everyone! Thank you for the input, I am using TMAP(modified BWA) and BWA. So I have not used bowtie 2 before but it is a splice aware mapper correct? So I have 2 votes for using a splice aware vs a non-splice aware. Correct?

[GenoMax]

Add BBMap to the list if you are looking at alternates.

[Brian Bushnell]

Hi everyone! Thank you for the input, I am using TMAP(modified BWA) and BWA. So I have not used bowtie 2 before but it is a splice aware mapper correct? So I have 2 votes for using a splice aware vs a non-splice aware. Correct?

No, Bowtie2 is not splice-aware.

Add BBMap to the list if you are looking at alternates.

+1

[SylvainL]

Yep, actually my mistake. I wanted to mention STAR (based on Bowtie2) which is splice-aware...
You can also use TopHat but, to my knowledge, bwa is not splice-aware...

[westerman]

... You can also use TopHat but, to my knowledge, bwa is not splice-aware...

What do those two sentence parts have to do with each other? Tophat is splice-aware and uses bowtie2 as its mapping engine.

[SylvainL]

skbrimer mentioned he used bwa, which is not splice aware. And I spoke about TopHat (and STAR in a previous answer) which are both splice-aware... I did not mean that TopHat was based on bwa...