Sam’s Notebook: Samples Submitted – Robertos C.gigas DNA for Whole Genome Bisulfite Sequencing (Genewiz)

Sent Roberto’s 12 designated DNA samples for whole genome bisulfite sequencing (WGBS) to Genewiz. They will do all bisulfite conversion, library prep and sequencing (Illumina HiSeq; 2x150bp; 30x coverage).

Also sent two samples prepared by Shelly (Panopea generosa) and two samples prepared by Yaamini.

Here’s the list of samples:

Roberto

  • 0501
  • 3501
  • 5201
  • 5901
  • 0502
  • 3502
  • 5202
  • 5902
  • 0530
  • 3530
  • 5230
  • 5930

Shelly

  • Tank3-15_16
  • Tank2-025_026

Yaamini

  • YRVA
  • YRVL

Turnaround will probably be around 30 days.

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Kaitlyn’s notebook: oyster seed proteomics comparisons

Comparing ASCA and clustering identified proteins

I modified and reran the clustering code using the technical replicate averages. The code and other plots can be found here. I used the proteins ASCA identified as being affected by temperature.

This is the code I used to compare the analyses. A total of 25 proteins were common between clustering and ASCA analysis. ASCA identified 113 different proteins while clustering identified 8 other proteins. It would be interesting to see how manipulating the dendrogram height cutoff value during clustering affects the latter number (I used a cutoff of 250).

Here is a heatmap of the similar proteins (note the first column is Day 0 followed by 23C days 3-13 and finally 29C days 3-13):

Venn Diagram of 23C and 29C

I’m working on making a better venn diagram in R with just the silo 3 and 9 proteins. Here is a quick and dirty venn digram, but I’m trying to usethe VennDiagram package to improve it.

Note that all 0 values were changed to 0.100 in the technical replicate averages. I removed all values less than 0.600 as these would be undetected in the silo because of the change (there are 6 days of data since were are excluding day 15 because of the temperature malfunction).

This is obviously still a work in progress to create a better venn diagram.

Yaamini’s Notebook: WGBS Samples Part 2

Continuing isopropanol precipitations

While thinking about my WGBS sample preparation last night, I realized that I don’t have enough of my low pH sample! I had a sample concentration of 20.8 ng/µL, but only 14 µL of sample after the Qubit. This means that sample volume was less than 500 ng and unusable for sequencing. After talking to Shelly, I did the following:

Isolate new samples

I needed at least 200 ng of sample to reach the 500 ng minimum. I took 6.3 µL of 9-T2 and 5.5 µL of 10-T3, added them to a new tube, then vortexed the new pooled sample. I put the pooled sample back in the fridge until I needed it.

Reprecipitate supernatant

I took the isopropanol supernatant I saved and added 5.31 µL more sodium acetate and 15 µL of isopropanol. I then spun the tube for 30 minutes at 12000 rpm and 4ºC. When I went to pipet the supernatant off, I couldn’t see a white pellet. I took off the supernatant and disposed of it, leaving a little bit of liquid at the bottom. For an ethanol wash, I added 90 µL of 75% ethanol and spun the tube for 10 minutes at 12000 rpm and 4ºC. Once again, I couldn’t see a clear pellet. I took off as much ethanol as I could and warmed the tube for 2 minutes on a heat block set to 37ºC. I took a little bit of remaining liquid and added it to the samples to the 200 µL of pooled samples I made prior.

With the ethanol wash supernatant, I added 2.5 µL of sodium acetate and 10 µL of 75% ethanol. I then put the tube in the -80ºC for an hour. After the incubation, I spun the tube for 30 minutes at 12000 rpm and 4ºC. Just like the ethanol supernatant, I couldn’t see a clear pellet. I did an ethanol wash with 90 µL of 75% ethanol then spun the tube for 10 minutes at 12000 rpm and 4ºC. After removing the supernatant, I set the tube on the 37ºC heat block for 2 minutes. I used the 200 µL of pooled samples to wash out any DNA stuck to the side of the ethanol wash supernatant tube. I had to run to class, so I left the tube to incubate at room temperature for a few minutes.

Precipitate supernatant and sample combination

Shelly was nice enough to measure my sample concentration! Here’s what she did:

Before I combined the two tubes, one looked particularly cloudy, which is odd. So I combined the samples and precipitated again (40uL sample + 28uL isopropanol + 4 uL 3M NaOAc). Vortexed well. Spun for 30 min @4C. Pipetted off supernatant. There was a white pellet, which probably shouldn’t be so white. I think that indicates a lot of salt. So I did 2x 1mL washes with 75% ethanol each spinning for 30 min. The pellet still seemed pretty white. Then let dry at room temp open cap for 15 min. Then resuspended in 11uL EB (Qiagen, from Sam’s bench), and it was much less cloudy. Then let sit at room temp for 30 min before reading concentration.

The final concentration was 64 ng/µL in 10 µL. I have more than enough sample to send for sequencing!

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Yaamini’s Notebook: WGBS Samples

Preparing C. gigas DNA for WGBS

We’re planning on sending samples for whole genome bisulfite sequencing, and Steven asked me prepare two pooled samples with C. gigas broodstock DNA I extracted. These samples needed to have at least 500 ng of DNA and a concentration of 20 ng/µL. I decided to make 2 pooled samples: one for the low pH group and one for the ambient pH group. For each pooled sample, I used two stage 0 oyster samples (sexually undifferentiated) with the highest yield. This way, I could hopefully use the samples for sequencing again, and I’d have a baseline for DNA methylation without sex effects. Since each pooled sample needed 500 ng, I obtained 250 ng of DNA from each individual sample.

Low pH sample (WGBS-YRVL 2/4)

9-T2: 15.9 ng/µL, 795 ng total in 50 µL. I used 15.8 µL of this sample.
10-T3: 18.3 ng/µL, 915 ng total in 50 µL. I used 13.7 µL of this sample.

The total volume of this pooled sample was 29.5 µL, and the concentration was 17 ng/µL. This was too dilute for sequencing, so Shelly helped me do an isopropanol precipitation to concentrate the sample, modified from her protocol. I added 20.65 µL of isopropanol to the sample (70% of original sample’s volume) and 2.95 µL 3M sodium acetate (10% of original sample’s volume). While I added the reagents to the sample, Shelly placed the centrifuge in the 4ºC fridge. We spun the sample for 30 minutes at 12000 rpm at 4ºC. The DNA was pelleted out and was white because of the salts. I removed the supernatant and saved it in a different tube, just in case (YRVL sup 2/4). I then added 75% ethanol and spun the sample and ethanol for 10 minutes at 12000 rpm and 4ºC. After the ethanol wash, I once again remove and saved the supernatant (YRVL EW 2/4). I kept the sample on a heat block at 37ºC for 2 mintues to dry the sample. While the sample was on the heat block, I also warmed the elution buffer from the E.Z. DNA kit. I then added 15 µL of the elution buffer to my sample and used the Qubit to measure the concentration. My final sample concentration was 20.8 ng/µL.

Ambient pH sample (WGBS-YRVA 2/4)

11-T4: 15.7 ng/µL, 785 ng total in 50 µL. I used 16.0 µL of this sample.
UK-01: 63 ng/µL, 3150 ng total in 50 µL. I used 4.0 µL of this sample.

My final sample concentration was 25 ng/µL in 20 µL. I did not need to modify this sample.

I saved both samples in the fridge so we can send them off for sequencing when UW operations aren’t affected by snow.

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