Grace’s Notebook: Closer to having some pooled libraries!

Sam extracted RNA using the Quick DNA/RNA Microprep Plus Kit (ZymoResearch) (post). I don’t think there is currently enough RNA in the libraries to reach the requiremets for NWGC… details in post.

Today I quickly joined his qubit result file with the hemolymph sampling data (hemo_table.csv) in R.

The resulting file: hemo_qub-for-libs.csv

The goal is to have 12 libraries accounting for each infection status and temperature treatment. Each pooled library needs to have at least 1000 ng of RNA in a sample of ~50ul. (GitHub Issue #184)

Pooled libraries to create:

Library Number Sample Day Infection status Temp trtmnt
1 9 0 NA
2 9 1 NA
3 12 0 Amb
4 12 1 Amb
5 12 0 Cold
6 12 1 Cold
7 12 0 Warm
8 12 1 Warm
9 26 0 Amb
10 26 1 Amb
11 26 0 Cold
12 26 1 Cold

*Note: the temperature treatments didn’t start until after the Day 9 sampling day.

As of now, I don’t think the pooled samples would have enough RNA to meet the requirements… for example, the samples that fall under Library 1 add up to 79.9 ng/ul.

I may need to check this again when I’m not sick.

from Grace’s Lab Notebook

Shelly’s Notebook: May 8, 2019 Geoduck broodstock hemolymph DMR calling

Running methylkit:

I had planned to run MethylKit on Emu’s RStudio web interface to find DMRs in the WGBS hemolymph. In reviewing a previous MethylKit R script, I realized I need sorted bam files, which my initial bismark mox script didn’t include. So I submitted a new mox job to sort my bams. Once the job finishes (after Mox maintenance), I will copy the sorted bams over to gannet and then on to Emu so I can run the Rstudio web interface (a little painful, but getting there!).

Running methylpy

So I thought it might be easier to install methylpy on the server and do DMR calling with this python program instead of with MethylKit in R. Turns out installing this on Mox was too challenging as I kept getting permission denied messages. So I tried installing it on Ostrich which turned out to also be very challenging. Finally, I got it installed and functioning after correctly installing GSL. I am currently running methylpy DMRfind on Ostrich.

from shellytrigg