DESEQ2 is not normalizing HTSEQ counts for some samples

Question: DESEQ2 is not normalizing HTSEQ counts for some samples

11 months ago by

I have RNAseq HTSEQ count data for 3 individuals collected at 3 time points. I would like to perform count normalization across all 3 time points for each individual separately using Galaxy DESEQ2. So far I find that DESEQ2 is providing normalized counts for some samples but it outputs the original raw HTSEQ counts for others (2 out of 9 samples consistently have this issue). Has anybody encountered this before? Thanks, Sergey

normalization htseq rna-seq deseq2 htseq_count • 607 views

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modified 11 months ago by Jennifer Hillman Jackson ♦ 25k • written 11 months ago by sergey.yegorov • 0

Hi there, I am dealing with the same issue with my data, one of my replicates is not normalizing in DEseq2 even though all replicates were analyzed with the same settings. I was curious if you were about to resolve this issue or could provide more information. What I've tried so far: -Remapping the original .fasta file -Doing htseq-count again from the new alignment (counts matched the first attempt) -Using DEseq2 with all replicates plus this new one; found that this replicate was still the only one not normalizing.

ADD REPLY • link written 6 months ago by kjh318 • 10

11 months ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

For your case, the experimental design may need some parameter tuning or a parameter intended to be set was missed. Double check how you set this up against the tutorials linked below to determine why the results differ between samples. I would also suggest reviewing the upstream jobs (mapper - HISAT2? and htseq_count itself) to ensure all data was processed the same way.

Galaxy tutorials for RNA-seq can be found here along with example data: https://galaxyproject.org/learn/

Those in the first group have been modified to work with the tools available at https://usegalaxy.org, for example: https://galaxyproject.org/tutorials/rb_rnaseq/

And this tutorial from the GTN, also linked from the page above, contains the same basic data from the usegalaxy.org tutorial, and also explains normalization methods in detail: https://galaxyproject.github.io/training-material/topics/usegalaxy/tutorials/rb-rnaseq/tutorial.html

Thanks! Jen, Galaxy team

ADD COMMENT • link written 11 months ago by Jennifer Hillman Jackson ♦ 25k

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