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I am trying to analyze an RNA-seq experiment from an organism with no genome sequence (the marine snail Ilyanassa), and I have been trying to use cufflinks to do this. I used a transcriptome assembly of solexa and 454 reads (made with velvet and mira) to make an index for the reference sequence for tophat.
My first question is whether there is anything special that I need to be doing since I am feeding it spliced transcripts? It seems to have worked at the gene level, but I would just like to be sure that I am not violating some assumption.
Second question, about cuffdiff specifically: I have an experiment and control lanes of 100 bp solexa reads (30 mil each). I made a joint .gtf file in tophat and cufflinks using all the reads from both lanes, then I measured each set of reads separately against this file using cuffdiff. I only got around 1700 sequences in the 0_1_gene_exp.diff file, even though there are ~192000 transcripts in the joint gtf file. The 0_1_gene_exp.diff file contains transcripts with hits in only one or neither (or both) and transcripts with too few to test, and transcripts that are tested but not significant. I can't figure out what I am missing here.
Thanks in advance for any advice!
Dave
My first question is whether there is anything special that I need to be doing since I am feeding it spliced transcripts? It seems to have worked at the gene level, but I would just like to be sure that I am not violating some assumption.
Second question, about cuffdiff specifically: I have an experiment and control lanes of 100 bp solexa reads (30 mil each). I made a joint .gtf file in tophat and cufflinks using all the reads from both lanes, then I measured each set of reads separately against this file using cuffdiff. I only got around 1700 sequences in the 0_1_gene_exp.diff file, even though there are ~192000 transcripts in the joint gtf file. The 0_1_gene_exp.diff file contains transcripts with hits in only one or neither (or both) and transcripts with too few to test, and transcripts that are tested but not significant. I can't figure out what I am missing here.
Thanks in advance for any advice!
Dave