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Old 01-11-2016, 12:44 PM   #1
skbrimer
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Default Split N Trim RNAseq data

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?
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Old 01-11-2016, 07:31 PM   #2
Brian Bushnell
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It seems prudent to map with a splice-aware aligner, just to be on the safe side.
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Old 01-12-2016, 12:50 AM   #3
SylvainL
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Hi,

simply tell us with mapper you used. If you used bowtie2 it should be fine...
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Old 01-13-2016, 08:16 AM   #4
skbrimer
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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?
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Old 01-13-2016, 09:37 AM   #5
GenoMax
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Add BBMap to the list if you are looking at alternates.
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Old 01-13-2016, 09:37 PM   #6
Brian Bushnell
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Quote:
Originally Posted by skbrimer View Post
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.

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Originally Posted by GenoMax View Post
Add BBMap to the list if you are looking at alternates.
+1
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Old 01-14-2016, 12:44 AM   #7
SylvainL
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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...
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Old 01-19-2016, 01:03 PM   #8
westerman
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Quote:
Originally Posted by SylvainL View Post
... 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.
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Old 01-20-2016, 02:06 AM   #9
SylvainL
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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...
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