This week we talk about transcriptome assembly (with our new RNAseq data from NWGC!!) and next steps. Also, we decide that Qiagen RNeasy Kit works pretty well for extracting RNA, and will be used to create the remaining libraries for the project.
This week Pam tells us about her recent experience on a survey on the Bering Sea, and we discuss our new plan.
The original RNA extraction method using RNAzol RT resulted in low RNA yields, as well as contaminated samples, likely due to excess salts. We have submitted a pooled sample to the NWGC for library prep and sequencing. The pooled sample consists of samples from day 26 (samples were taken in triplicate) from cold and ambient infected and uninfected treatments. This library will be used for gene discovery.
As we continue investigating better methods for RNA extraction, we will create libraries that will be used for comparison between treatment groups.
This week we discuss our plan of having three pools sequenced and next steps going forward. Three pools for the current three libraries are as follows:
- uninfected from Day 9 (before temperature treatment began)
- infected from Day 9 (before temperature treatment began)
- “MasterPool” : 10 samples. One sample per each of the following treatments:
- uninfected, day 12, cold
- infected, day 12, cold
- uninfected, day 12, ambient
- infected, day 12, ambient
- uninfected, day 12, warm
- infected, day 12, warm
- uninfected, day 26, cold
- infected, day 26, cold
- uninfected, day 26, ambient
- infected, day 26, ambient
This week I speak with Genevieve Johnson, a MS student at University of Alaska, Fairbanks. We talk about her thesis work on population genetics of Alaskan Tanner Crabs, her experience taking crab samples on a vessel, and her thoughts on her future.
This week is Part 2 of FAQ with Pam. We talk about:
- Mass die-off in warm treatment crabs
- Crab transport
- terminal molt
- Signs vs symptoms of BCS (Bitter Crab Syndrome/Bitter Crab Disease)
This week we discuss R scripts for combining files, qPCR results for infection status, and next steps.
In this episode, Pam answers some questions I and others have on the issue and project.