Planning for WGBS

Last year I extracted C. gigas broodstock DNA and sent off pooled gonad samples for WGBS. Since I don’t have enough DNA to do MBDBS with these samples, I’m seriously considering WGBS. Based on this issue from Sam, Zymo WGBS sequencing for 10 samples should be affordable and doable. Plus, my previous samples were sequenced by Zymo so there would be consistency there!

After a chat with Steven, I decided to investigate if I can sequence 5 samples per treatment. I would need at least 500 ng and 20 ng/µL for each sample. I looked at this sample spreadsheet updated the amount of DNA I had left after creating the pooled samples. Then, I identified samples from each treatment that had at least 500 ng of DNA. I tried balancing extraction batches and maturation stage, choosing only female samples since we saw a female-specific carryover effect. I also looked at the histology blocks to see if there was any tissue left for paired RNA extraction! I felt good about the sample if more than half of the tissue was left for future extractions. Here are the samples with caveats if they exist:

Low:

• 02-T1
• 04-T3
• 06-T1: No tissue left for RNA extraction
• 07-T2
• 08-T2: Less than half of tissue sample left for RNA extraction
• 10-T3

Ambient:

• 11-T4
• UK-01: No more than half of sample left for RNA extraction
• UK-02
• UK-03
• UK-04: This sample has less than 500 ng, but I wanted another sample that was a maturation stage of 2 and not indiscriminate like the rest of the ambient samples. I posted this issue to see if there’s any wiggle room. According to Sam, there may not be but Zymo might be more flexible.
• UK-06: Less than half of tissue sample left for RNA extraction

My next steps are to see if I need to do any library preparation so these samples can be sequenced.

Going forward

1. Confirm Zymo quotes and finalize samples for WGBS
2. Double check DNA quality and concentrations
3. Prepare libraries (if needed)
4. Extract RNA from gonad samples