DESeq2-issues with input data
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3.4 years ago
vivek37373 • 0

I have a small rna dataset of 12 samples containing two replicates for specific conditions. For example,

CON1 ---> 30 million reads


Using the raw counts from these conditions reports only two DEG genes, which I guess because of the huge difference in coverage between replicates. Is there any possible way to use this data to screen significant genes. Any help, suggestions or ideas in this regard would be appreciated.

Cheers

deseq2 smallrna RNA-Seq • 912 views
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F2 is the only one that has a low read count compared to the others, but everything else is within a range that the normalisation should handle - and even F2 could be still ok. I don't have any literature reference for this. I guess its rather the high variance between replicates and only 2 replicates per condition. You should look at more diagnostic plots: clustering, PCA, where do the reads align to, ratio of alignments ...

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Without knowing experimental details such as the differences between the conditions, your experiment is probably underpowered in terms of replicates. n=2 per condition is low. Replication number is more important than sequencing depth.

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I completely accept your point. However, the sequencing performed was limited to funds.

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Can you follow up to the points by Ido Tamir, too? - they are quite relevant, in particular, this:

You should look at more diagnostic plots: clustering, PCA, where do the reads align to, ratio of alignments ...