We have paired-end Illumina RNASeq reads and we are working with a non-model organism with no reference genome. We have a working composite for a protein sequence that includes every exon we have found via cDNA. We have 6 muscle types with some triplicates and want to see how many times 4 specific exons that look to be alternatively spliced are present in each muscle type.
For example, muscle type a has this exon expressed 46% while muscle type b only expresses this exon 12% of the time.
I figured I could extract the exon sequences individually and then align within HISAT2 and feed into stringtie for abundance counts. That way we are only including the transcripts aligned to that specific exons. I'm not looking for differential expression, only a number of how many times this exon is found within the muscle type's transcript file.
Following stringtie I was thinking of putting the HISAT2 BAM file and GTF file (from stringtie) in something like htseq-count or featureCount. Is something like this doable? It's really only for a figure that we're trying to construct showing how many times a transcript within a muscle type maps to a certain exon within one protein sequence.
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You should first build transcripts using
trinity(https://github.com/trinityrnaseq/trinityrnaseq/wiki ) with all of the data together. Once you do that you should be able to align your data back to those models and get counts you need.If you do have gene models (transcripts, you have not mentioned so clearly) then you could use salmon/kallisto to do alignment free estimation of expression.oh yes, we do have the trinity transcripts- sorry. how do I select for a specific exon when I use kallisto with the cDNA composite and muscle trinity transcript file?
Sorry I overlooked the
exonlevel estimates part in your original question. If you just need exon level estimates (and already have exon sequences) can you not align to those and just count the reads that are aligning to each exon (assuming you have a strategy for multi-mappers i.e. random, discard, all places)?Well originally, we did do that. We aligned the transcript to the entire composite and then viewed it in IGV and counted the transcripts that aligned to it. But we were hoping that since each transcript file contains hundreds of thousands of transcripts there would be an easier to way to just get a number output rather than counting them individually