Hi,
I know this is a broad question, but I figured I would see if someone can give me even a little bit of help. I had some RNA sequencing done for me and I was interested to see if I could find some novel genes and/or some new isoforms. The species I work with isn't super well studies, but ensembl does have protein predictions so I do have that as a reference. I have had issues with these protein predictions in the past, and they haven't always been the most accurate predictions (this is probably due to the fact that I work with an odd species). Anyways, I have this RNA-seq data and I want to try and maybe find some novel genes or if the predictions are accurate, maybe some new isoforms for an already discovered gene. I was wondering if anyone had any suggestions on how to do this? I know the whole ballgown method is way out of date. I saw something about a new program call LeafCuter and another one called DEXSeq. I am just looking for the best program to use. I know these program can go out of date fast so I don't want to waste time on a less desirable program. Those two seemed kind of cool because you could use R to visualize the data.
I already did some DE on the data using Salmon and Deseq2 so I don't need any programs for that.
Thanks for the help!
Hi Kevin Blighe
I have one query and I think you help me to do it in a faster way.
I have analysed RNA seq data using Kallisto due to system limitation and the purpose was to only detect DEG, not any novel isoform discovery and sorted out a list of gene. Now I am interested in studying a gene in details but when I checked the same gene in human multiple isoforms are present but in my species, only a single isoform is annotated.
I have hypothesised that other isoform might be expressed in my species because at protein level I couldn't detect the protein (only possible when other isoforms are there because the epitope is missing in other isoforms in human) for that gene which is involved in the phenotype. So I just want to detect a novel isoform for that gene. can I take only the gene sequence and allign RNA seq data to detect novel isoform by any tools.
Thank you will appreciate your suggestion
Hi, so, you do not have sufficient resources to use a splice-aware genome aligner like STAR? Which transcriptome reference FASTA did you use for Kallisto? At the most basic level, you can add the cDNA of your isoform of interest to your reference transcriptome FASTA, and re-do the pseudo-alignment. Perhaps ask a new question if this is not answering your question.