TRINITY Pipeline

Well, there’s good news and bad news. The good news is that I don’t actually need to run the TRINITY pipeline to create a reference transcriptome - we already have some reference transcriptomes assembled! The bad news is…dang, that’s a lot of work down the drain. As a result, I’ve shifted towards the downstream analyses found in the sidebar of (the Trinity manual)[https://github.com/trinityrnaseq/trinityrnaseq/wiki].

General overview of next steps:

  • Select which transcriptomes to examine - not choosing crab- or hemat-only (DONE)
    • cbai_hemat_transcriptome_v2.0: contains all pooled libraries & individual crab samples.
      • Encountering problems with running it through Kallisto (elaborated upon below)
    • cbai_hemat_transcriptome_v3.0: contains all pooled libraries
  • Quantify transcript abundance with kallisto
    • Build index
    • Quantify abundance in each library
  • Join with BLAST annotation tables in R or Bash

Kallisto Well, Kallisto must’ve run into Hera, because it’s definitely having some problems.

I successfully built an index for transcriptome 3.0 (although it took around 36 hours), but my computer refused to build an index for transcriptome 2.0 - not enough RAM. I’m looking into getting Mox access (which I set aside after figuring out I wouldn’t need it to create a reference transcriptome), but for the time being, I’m planning to move forward with my analysis using transcriptome 3.0, and come back to 2.0 when I can.

Again, I’m performing 4 total comparisons between different groups of infected crab

  • Library 2 vs 4 (Day 2 low-temp vs. Day 2 high-temp)
  • Library 2 vs 6 (Day 2 low-temp vs. Day 0 ambient)
  • Library 4 vs 6 (Day 2 high-temp vs Day 0 ambient)
  • Library 8 vs 10 (Day 2 all temps vs. Day 17 all temps)