Question: Consequences of assembling strand-specific RNAseq as if it was not strand-specific
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gravatar for RNAfan
3.0 years ago by
RNAfan10
RNAfan10 wrote:

Hi,

Recently I worked on assembling a transcriptome of a non-model organism. I was told the RNA-seq data was not strand-specific, and I assembled the transcriptome with Trinity, using the not strand-specific option.

It turns out that the reads are actually strand-specific. I would like to know what are the consequences of keeping the current transcriptome (in other words, of analyzing strand-specific data as not strand-specific).

My study organism is a vertebrate, so I don't think the genome is particularly compact. The main use of this transcriptome was to guide a genome annotation process. We are quite satisfied with the annotation derived from the current transcriptome.

I know that the best thing to do would be to redo the analyses, but this would be time consuming and would require a lot of computing resources, that we don't have right now. I guess I am looking for reasons to convince my boss that this is what we should do.

Thanks!

trinity rna-seq assembly • 963 views
ADD COMMENTlink modified 3.0 years ago by Biomonika (Noolean)3.0k • written 3.0 years ago by RNAfan10
0
gravatar for jotan
3.0 years ago by
jotan1.2k
Australia
jotan1.2k wrote:

I doubt that strand-specificity will have a big impact on your results.

But, you absolutely have to re-do the analysis. It would be irresponsible to not correct a technical error once you know it's there.

ADD COMMENTlink written 3.0 years ago by jotan1.2k
0
gravatar for Biomonika (Noolean)
3.0 years ago by
State College, PA, USA
Biomonika (Noolean)3.0k wrote:

You likely got worse assembly (than what you would get, had you specified strand-specifity). You can evaluate how much worse - for each transcript, count number of reads mapping in both directions, looking at which strand first read in a pair maps (you can find details how to do this in these posts: https://www.biostars.org/p/87097/). Since antisense transcription is very rare, this can either reassure you that your assembly is fine or give you evidence that you should redo it.

ADD COMMENTlink modified 3.0 years ago • written 3.0 years ago by Biomonika (Noolean)3.0k
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