I completed one RNA seq analysis using SRA datasets of a plant sample infected and non-infected. After DESeq2 output for DEGs, I wanted to annotate the IDs according to annotation file. I uploaded the annotation file and run “Annotate DESeq2/DEXSeq output tables”. However, the output result file did not include any annotation but only NA for all the DEGs. I am using reference genome G.max Wm82.a4.v1. Used the same version files for assembly and gff3 annotation file.
I am not sure what I am doing wrong here. I tried to check the IDs if they have annotation in the file and found that their annotation is present but the output of this tool doesn’t show any annotation except NA for all databases.
Welcome, @Amjad
Check under the Advanced Options for this tool. The default attributes the tool is using might need to be changed to match your GFF3 annotation’s actual attributes.
Hi Jennaj, thank you for prompt response. Could you please be more specific or is there some guide for understanding the attributes?
Hi @Amjad
Find an online specification for the GFF3 data – where you sourced the data should have that. Then review the annotation you have in that context. Some will be in the 3rd column and some in the 9th if the format is standardized from that source.
Then, expand the advanced settings for this tool. Each section is labeled, and is at default appropriate for a (most?) GTFs but not for GFF3. Then, adjust the settings to match your data, or decide to use different annotation data. Not all annotation works well with all tools.
I added a few tags to this topic – but you can also just search the forum with the datatypes to see what worked for others. None of this is exact, and understanding what data attributes are used will matter when interpreting the outputs.
Many prior QA will point to this general FAQ Working with GFF GFT GTF2 GFF3 reference annotation