Grace’s Notebook: September Goals

2015 Oysterseed Project:

  • Figure out Skyline DIA with Emma (plan for 9/6/18)
  • Analyze 2015 Oysterseed data

Crab Project:

  • Lyophilizer is fixed –> use this along with Tri-Reagent on selected pelleted and supernatant samples
  • Continue to learn more background information on C. bairdi, Hematodinium, Bitter Crab disease, etc.
  • Practice qPCR to test shellfish primers; learn more about qPCR

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Sam’s Notebook: DNA Methylation Analysis – Olympia oyster BSseq MethylKit Analysis

0000-0002-2747-368X

NOTE: IMPORTANT CAVEATS – READ POST BEFORE USING DATA.
I’m posting this for posterity and to provide an overview of what (and whatnot) to do. Plus, this has a good R script for using MethylKit that can be used for subsequent analyses.

The goal of this analysis was to compare the methylation profiles of Olympia oysters originating from a common population (Fidalgo Bay) that were raised in two different locations (Fidalgo Bay & Oyster Bay).

An overview of the experiment can be viewed here:

I previously ran all of Olympia oyster DNA methylation sequencing data through the Bismark pipeline, and then processed them using the MethylKit R library.

First mistake (Bismark):

  • Trimmed FastQ files “incorrectly”.

Bismark provides an excellent user guide and provides a handy table on how to decide on trimming parameters, but I mistakenly trimmed these according to the recommendations for a different library preparation technique. I trimmed based on the Zymo Pico-Methyl Kit (which was used for the other group of data that I processed simultaneously), instead of the TruSeq library prep.

So, “incorrectly” isn’t necessarily the proper term here. The analysis can still be used, however, it’s likely that the excessive trimming results in reducing sequencing coverage, and, in turn, making the downstream analysis result in a highly conservative output. Thus, the data isn’t wrong or bad, it is just very limited.

And, this leads to the second mistake (Bismark):

  • Bowtie alignment score too strict

There’s a bit of a weird “battle” between Bismark and bowtie2. Bismark uses bowtie2 for generating alignments, but bowtie2’s default cutoff score overrides Bismark’s. So, to adjust the score value, you have to explicitly add the scoring parameters to your Bismark parameters during the alignment step. I did not do this.

Again, it’s not wrong, per se, but leads to a significantly limited set of data in the final analysis.

The data were analyzed based on a minimum of:

  • 3x coverage
  • 25% difference in methylation

Sam’s Notebook: Transcriptome Assembly – Geoduck RNAseq data

0000-0002-2747-368X

Used all of our current geoduck RNAseq data to assemble a transcriptome using Trinity.

Trinity was run our our Mox HPC node. Specifically, I had to use just a single node with 500GB of RAM. Trinity could not run with much less than that. Initially, I attempted to run with two nodes, but our smaller node (120GB) ended up limiting the available RAM (the system only uses the RAM available on the smallest node; it cannot combine RAM or dynamically allocate computing to a node with larger RAM when needed) and Trinity consistently crashed due to memory limitations.

Reads were trimmed using the built-in version of Trimmomatic with the default settings.

SBATCH script:

Due to the huge number of input files, I won’t post the entire script contents here. Instead, here’s a snippet of the script showing the commands used to start the Trinity run:

  #!/bin/bash ## Job Name #SBATCH --job-name=20180829_trinity ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=30-00:00:00 ## Memory per node #SBATCH --mem=500G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite@uw.edu ## Specify the working directory for this job #SBATCH --workdir=/gscratch/scrubbed/samwhite/20180827_trinity_geoduck_RNAseq # Load Python Mox module for Python module availability module load intel-python3_2017 # Document programs in PATH (primarily for program version ID) date >> system_path.log echo "" >> system_path.log echo "System PATH for $SLURM_JOB_ID" >> system_path.log echo "" >> system_path.log printf "%0.s-" {1..10} >> system_path.log echo ${PATH} | tr : \\n >> system_path.log # Run Trinity /gscratch/srlab/programs/trinityrnaseq-Trinity-v2.8.3/Trinity \ --trimmomatic \ --seqType fq \ --max_memory 500G \ --CPU 28 \  

Despite the naming conventions, this job was submitted to the Mox scheduler on 20180829 and finished on 20180901.

After job completion, the entire folder was gzipped (the following method of gzipping is SUPER fast, btw):

 tar -c 20180827_trinity_geoduck_RNAseq | pigz > 20180827_trinity_geoduck_RNAseq.tar.gz

Yaamini’s Notebook: September 2018 Goals

T-24 days until classes…

It’s peak grind time.

August Goals Recap:

Manchester:

  • I addressed all comments from Sam and Laura, and all minor comments from both reviewers and George.
  • I’m almost done revising my discussion, which is the last step for reviewer comments

Gigas Broodstock:

  • I tested the DNA extraction protocol with the Genome Sciences Thermomixer a BUNCH and it didn’t really work (trial 1, trial 2, trial 3).
  • Drafted a plan for successful extractions without the Thermomixer! Hopefully that protocol will be successful

Virginica:

  • Nothing got done. Reviewer comments and protocol testing consumed my life.

September Goals

Manchester:

  • FINISH ADDRESSING REVIEWER COMMENTS AND SEND THE REVISED MANUSCRIPT TO JSR!

Virginica

  • Finish gene enrichment analysis
  • Update methods section
  • Write results
  • Outline discussion
  • Present at PCSGA!

Gigas Broodstock:

  • Test DNA extraction protocol
  • Extract broodstock DNA

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Kaitlyn’s notebook: DNA, labeling and cleaning

On Sunday I assisted Yaamini with DNA extractions from blocks. I scraped tissue from I’m blocks in the scheme shown below and added about 0.02g of tissue to corresponding tubes (T#-TT and T#-V2). Our main focus was testing the heat block and vortexing combination since the thermomixer was not available. Yaamini showed me how to use the Qubit since I’ve only ever used a Nanodrop. The vortex-heatblock combo appeared to work and we had some high levels of DNA! However, we need to use the bioanalyzer to ensure that the DNA was not sheared too much before we begin DNA extractions from the broodstock samples.

I also helped clean up the lab in preparation for Emily and Micah’s sampling. I bleached the stainless countertop where I think it will be best to dissect the young Olympia oysters. The lab inventory is updated with anything I put in drawers or moved and I made sure to wash all of the beakers and tools in the sink.

Today, I continued to label tubes. I’ve finished 455 of the tubes which is all of the 2.0ml twist top in the lab. The extra tubes should come I. Later today or tomorrow so o can finish the rest then.