I am hoping someone might be able to help me trouble shoot some issues I am having building an assembly which I would like to use as a reference for DE analysis. As the title states, I am using trinity and I am getting way too many transcripts. Before I go any further I will state that I have read the article "There are too many transcripts, what do I do?" but I think my problem is a bit beyond the scope of that post.
I am trying to build a de novo transcriptome assembly using pooled samples of sea urchin larvae (four samples representing one sample per four treatment levels). I am getting around 800,000 transcripts where I would normally expect to get around 70,000 to 100,000 for similar data sets. I know that at least part of the issue is that I have lots of duplicate genes/isoforms due to the nature of the samples (many pooled individuals), however this still seems extreme. I have tried collapsing my assemblies using Grouper and CD-HIT but the assemblies are so large that collapsing still produces very large assemblies. Does anyone have any suggestions?
Thanks.
It may be of interest to tell us some information about how much data went into this analysis, size of the genome and how you pre-processed the data.
Out of curiosity is there a specific reason to do a de novo assembly? Sea urchin genome has been available for over a decade and has a defined transcriptome (for S. purpuratus, available from Echinobase, NCBI or Ensembl). You could use this known transcriptome to weed out spurious transcripts from your assembly.