Question

DEG with low counts

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Rajat • 0

@dbe39373

Last seen 7 months ago

United States

Hi

I am currently analyzing RNASeq data, having used HISAT2 for mapping and StringTie for quantification. I've proceeded to use DESeq2 for differential expression analysis, but I've encountered an issue where many of the differentially expressed genes (DEGs) identified are lowly expressed according to the count matrix. Could this suggest a potential error in my analysis process?

I would greatly appreciate any guidance or suggestions you might have to help address this issue.

Best Rajat

DESeq2 • 546 views

ADD COMMENT • link updated 7 months ago by Michael Love 43k • written 7 months ago by Rajat • 0

score 0 · Answer 1 · 2024-04-15

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vincent.carbonnier • 0

@d215cb6b

Last seen 1 minute ago

France

Hi,

Did you have a terminal input and output copy? I think the error could comes from the GFF/GTF file. Either an organism or genome version error.

Best,

Vincent

ADD COMMENT • link 7 months ago vincent.carbonnier • 0

score 0 · Answer 2 · 2024-04-15

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Michael Love 43k

@mikelove

Last seen 10 hours ago

United States

Can you include plotCounts for a gene? or plotMA for the dataset?

ADD COMMENT • link 7 months ago Michael Love 43k

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Dear Dr. Michael

Thanks for you quick reply. I appreciate it. Attached is the plotcounts for one gene. I am experiencing like this in other genes as well. Also I am providing the log2fold change at different timepoints. geneID SUS_C vs SUS_06 SUS_C vs SUS_24 SUS_C vs SUS_48 TOL_C vs TOL_06 TOL_C vs TOL_24 TOL_C vs TOL_48 Glyma.05G004300 -19.32637569 -40.69880385 -2.467047927 36.75696216 37.9392758 39.54766649 enter image description here

Here in the figure, expression in one replication in TOL_6 and TOL_24 is making gene significantly upregulate with adj pvalue significantly less than 0.01. So, this is making me worry if I am analyzing my data wr

ADD REPLY • link 7 months ago Rajat • 0

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These counts are too low, only a few samples above a count of 10. So for typical RNA-seq data that is underpowered to call differences.

It's hard to distinguish from

plot(rep(1:8, each=3)+rnorm(24,0,.1), rnbinom(24, mu=3, size=.1))

enter image description here

ADD REPLY • link 7 months ago Michael Love 43k