Hi, everyone!
I have been using Cuffdiff to determine differential gene expression in Serratia marcescens. I notice that multiple genes shared the same transcript, giving the same FPKM values instead of each individual gene in the final testing. Therefore, I compared the genome and the annotation file (gff3 format) which is downloaded from NCBI by Artemis. I found that all the genes that overlapped seem to be merged as one gene. Is this a known issue with Cuffdiff and is there a way to solve it in order to get the FPKM for individual gene even though they are overlapped?
Thanks for your help! :)
It happens because the map coverage between these two genes are continuous (it can happen with 2 or more genes) and cuffdiff can't differentiate them. If you have high transcriptome coverage and genes very close to each other, you'll see this lot! Other reason can be the wrong gene prediction during structural annotation. Some gene predictors split real genes in two, or more sometimes. Especially if you used a small number of predictors.
To avoid this intergenic mapping, use the option -T on tophat command line.
Tophat manual:
"The following options in this section are only used when the transcriptome search was activated with -G/--GTF and/or --transcriptome-index.
-T/--transcriptome-only Only align the reads to the transcriptome and report only those mappings as genomic mappings."
Cufflinks/Cuffdiff might not be the best option to analyze bacterial RNA-seq. Maybe you can try other options like EDGE-pro http://ccb.jhu.edu/software/EDGE-pro/index.shtml
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