Laura’s Notebook: September, 2019 Goals

Review of last month’s goals

QuantSeq:

  • Quantify remaining RNA with Qubit – complete
  • Assess RNA quality via Bioanalyzer on subset of samples – complete
  • Concentrate dilute samples. Goal is 500 ng in 5 uL – not done
  • Acquire all materials for QuantSeq library prep – complete
  • Test protocol on 8 samples (“extra” samples). – in progress

Degree:

  • Identify and reach out to potential new committee members – complete. GSR is Jen Ruesink!
  • Read Jackie’s stack of papers, take notes and organize using Evernote – in progress!
  • Get study lists from Rick & Steven – in progress. Received from Rick

Manuscripts:

  • Finish simplifying O. lurida temperature/food methods, results and discussion – complete, sent to Steven for review
  • sBegin revising, or at least organize thoguhts on, Polydora MS as per discussion with Chelsea & Julieta – not started!

PCSGA:

  • Review presentation from Aquaculture 2019 for PCSGA. – in progress
  • Measure larvae from 2017 OA/T study – complete

## New Goals

### QuantSeq

  • Troubleshoot issues with trial run :/
  • Make headway on preparation of actual samples

### Degree:

  • Continue reading to prep for Quals – need to pick up speed
  • Get reading list from Steven
  • Meet with Jen
  • Meet with Krista & Mac re: GRIP
  • Can I submit dissertation proposal?

Manuscripts

  • Oly temp/food
    • Revise methods -> discussion based on Steven’s feedback
    • Write intro
  • Polydora -> redraft (!)
  • Maybe get reviewer comments from Ecological Applications? (Not to-do, but something to anticipate)

### Other

  • NSA quarterly newsletter piece
  • Present @ PCSGA
  • Figure out Jackie’s scope calibration situation …

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Yaamini’s Notebook: September 2019 Goals

feels

It’s that part of the year where it’s too hot to think but you do your best anyways shrug

August Goals Recap

Virginica Gonad Methylation:

Other:

  • Registered for PCSGA
  • Dr. Lauren Buckley agreed to be my GSR!

September Goals

Finish. the. paper. ≥ PCSGA presentation > everything else

Virginica Gonad Methylation:

  • Write discussion
  • Write introduction
  • Update paper repository
  • Send paper to collaborators
  • Submit for the Epigenetics Special Issue

Gigas Gonad Methylation:

  • Run bismark alignment
  • Identify DML and DMG
  • Present preliminary findings at PCSGA

Other:

  • Register for WSN
  • Register for ASLO
  • Meet with Steven to discuss timelines for upcoming chapters and qualfying exams

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Yaamini’s Notebook: WGBS Analysis

Testing bismark parameters for WGBS data

We’re 18 days out from PCSGA, which means I should start analyzing my data! In February, I pooled C. gigas samples from ambient and low pH treatments for Whole Genome Bisulfite Sequencing (WGBS). I’m planning on processing the data to look at differential methylation between treatments so I can justify future sequencing.

Determining parameters

The first step in this process is aligning samples to a bisulfite genome with bismark. I started this Jupyter notebook to test different alignment stringencies with a subset of the data before running the full bismark pipeline. Sam already trimmed my files and prepared the bisulfite genome, so that’s two things I didn’t have to do! I downloaded the bisulfite genome from this link and trimmed samples from this link (names start with YRV). I would have run bismark without downloading first, but owl is super slow I’d rather just have them on genefish.

Once I had the files dowloaded, I referred to my C. virginica script for parameter testing. The last thing I wanted to do was confirm my files were paired and non-directional. I was pretty sure they were paired because there were two files for each sample. I wasn’t sure if they were non-directional. I posted this issue to confirm. After running different alignment stringencies, I found that score_min = L,0,-0.9 gave me about 61% alignment. Less stringent settings gave me 68% alignment, while more stringent settings gave me 47% alignment. I decided to stick with 61% alignment because it was the biggest jump between settings, and I didn’t gain much from less stringent settings.

Running bismark on Mox

Now that I had my alignment settings in order, I could run a full alignment on mox. I modified a previous script for bismark on Mox with my C. virginica samples to use with my C. gigas samples. I saved the revised script here. I used the following commands to move files over to Mox:

 rsync --archive --progress --verbose yaamini@172.25.149.226:/Users/yaamini/Documents/project-gigas-oa-meth/analyses/2019-08-30-Bismark-Parameter-Testing/*fastq.gz .#Transfer analysis files  
 rsync --archive --progress --verbose yaamini@172.25.149.226:/Users/yaamini/Documents/project-gigas-oa-meth/analyses/2019-08-30-Bismark-Parameter-Testing/Crassostrea_gigas.oyster_v9.dna_sm.toplevel . #Transfer bisulfite genome to Mox  
 rsync --archive --progress --verbose yaamini@172.25.149.226:/Users/yaamini/Documents/project-gigas-oa-meth/scripts/2019-09-03-Bismark.sh . #Transfer script to USER directory, not scrubbed directory  

To run the script:

 sbatch 2019-09-03-Bismark.sh  

It’s been a while since I last used Mox, so I encountered this error. Turns out --workdir is no longer used! I changed it to --chdir. I learned a new verison of bismark was installed, so I changed version 19 to 21 in the script. Once I made those changes, I reran the script! The job number was 1266351.

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Laura’s Notebook: QuantSeq Library Prep test-run

Katherine suggested I work through the library prep protocol with a few samples to practice and work out kinks. From her experience, the libraries she generated later in the game were of higher quaity. I’m generating 8 test libraries – this was her recommendation based on the heavy use of 8-channel pipettes.

Following the QuantSeq manual and tips from Katherine.

Quick reference for Libary Generation

image

Notes from this test run:

Started on 8/20/2019

Prior to beginning, I created programs on the PTC-200 DNA Engine Cycler. Programs are labeled “Quantseq-#”, with the # corresponding to step number in the QuantSeq manual.

first strand cDNA synthesis

  • Based on Katherine’s suggestions, I will generate 40 libraries at one, in 5 rows of 8 on a PCR plate. When adding solutions to each well, rather than using a single channel pipette and transferring solutions to individual wells, I’m going to use PCR strips, load enough of each solution into 8 wells (with a bit excess), and use a multichannel to distribute. This way I can hopefuly reduce time.
  • Between each step I need to quickly “spin down” my PCR plate. Our benchtop centrifuge has a minimum run time of 1 minute, and with the time it takes to accelerate and decelerate total time is ~4 minutes. The “quickly spin down” instructions do not define a centrifuge speed to use, so I set it to 3 rcf based on whatt is specified in the equipmentt list – “Benchtop centrifuge (3,000 x g, rotor compatible with 96-well plates”. To reduce the amount of time to spin down plates, I should either set the speed lower, OR simply start, then stop the centrifuge manually.

RNA removal

  • No notes.

second strand cDNA synthesis

  • SS1 and USS are indeed viscous; hold pipette in place when pulling volumes.
  • SAFE STOPPING POINT

Placed in -20C overnight.

Purificaion

(this is 1 of 2 purifications, dubbed “pre-PCR”)

  • As Katherine hinted, it’s important to have a magnetic plate that fits the PCR plates used. The plates we have just have rods – no wells for plates to hold plates in place, which doesn’t work. I ordered a magnet/plate from ebay to hopefully improve the process.
  • SAFE STOPPING POINT.

Placed in -20 on 8/21/19 until next step (qPCR assay).

qPCR assay for optimal # cycles

Performed on 9/3/2019

  • Created a custom qPCR protocol with Sam’s help –
qPCR Assay Mastermis Calcs
Item per rxn (uL) all rxns (inc. NTC) (uL) all rxns * 1.1
Number of samples 1 9
cDNA, diluted to 19uL 1.7 15.3 16.8
PCR mix (PCR) 7 63 69.3
P7 Primer (7000) 5 45 49.5
Enzyme mix (E) 1 9 9.9
2.5x SYBR Green I nucleic acid dye 1.2 10.8 11.9
Elution Buffer (EB) 14.1 126.9 139.6
Mastermix total vol 28.3 254.7 280.2
SYBR Green Calcs Volumes
Stock concentration 100
Desired concentratino 2.5
Total volume needed 2.5x 11.9
Volume 100x 0.2970
Volume DMSO 11.58
Dilution ratio (should be 1:40) 0.025
Final concentration 2.5

Results: Located on Owl, https://ift.tt/2A2cccd with today’s date.

No amplification :/ max RFU should be ~10×10^12, and my no-template control (NTC) has same curve as samples.

2019-09-03_ qPCR-assay-test-run

First troubleshooting step is to see if I actually synthesized cDNA – I believe I can use the Qubit for that. If yes, then I messed up the qPCR somehow (wouldn’t surprise me). Perhaps there is an issue with bubbles? Maybe BioRad settings needed be adjusted for sybr green?

I ordered a trial QuantSeq kit (n=4) for trouble shooting, and as a result spoke with the WA respresentative. Notes from our call:

  • If cDNA synthesis did not occur, then I most likely had contamination with organics or salts, which can inhibit 1st strand synthesis and cause cDNA to degrade when trying to degrade RNA.
  • The TurboDNase method might be an issue, since it did not include a column.
  • Using a cleaner column on existing RNA may do the trick. I did order one box of Zymo Cleaner-Concentrator (n=50), so could run all my samples through this column.
  • 500 ng of input RNA is not necesary (she said that “no one uses that much”). 100 ng should be adequate!

I will receive the trial kit tomorrow, and Kristy is connecting me with the tech support guy.

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