Project background

Experimental design

Adult C.virginica oysters were exposed to elevated pCO2 treatment for 30 days. Following this, gonads were sampled for gametes (sperm or eggs). Fertilization crosses were performed within treatments, resulting in ControlxControl and ExposedxExposed offspring. Offspring were reared in control conditions. 9 hours post-fertilization, zygotes were sampled, and 3 days post-fertilization, larvae were sampled.

Data

• WGBS for zygote and larvae offspring

Reference data from prior work

• WGBS for parent gametes (sperm and eggs)

• RNA-seq for parent gametes (sperm and eggs)

• Larval physiology (shell growth, shell morphology, survival)

Preliminary results (from prior work)

From partner studies, elevated pCO2 resulted in:

• no DEGs in parent gametes (did affect gene activity features like transcript expression per gene)

• differential methylation (parental sperm and eggs)

• parental exposure improved shell growth rate in larvae (improvements enhanced when offspring also reared in elevated pCO2)

Completed work

Official Github repo for the ceasmaller project: https://github.com/sr320/ceasmallr/tree/main

Official large-file storage (e.g., raw and trimmed reads, bismark output): https://gannet.fish.washington.edu/gitrepos/ceasmallr/output/

Steps already performed in the official repo:

1. Trimming. Raw WGBS FastQs were concatenated, trimmed using fastp, repaired if necessary using BBtools, and the quality-checked using FastQC and MultiQC. Details at ceasmallr/code/00.00-trimming-fastp.md

2. Alignment. Trimmed WGBS FastQs were aligned to the C. virginica genome using Bismark and Bowtie, then summarized using MultiQC. See details at ceasmallr/code/02.00-bismark-bowtie2-alignment.md

3. Deduplication. After reads were aligned to the C. virginica genome, they were deduplicated using Bismark to remove read duplication caused by PCR amplification during WGBS. See details at ceasmallr/code/02/10-bismark-deduplication.md

4. Methylation extraction. Bismark was used to call the methylation state of all sequenced cytosine positions. See details at ceasmallr/code/02.20-bismark-methylation-extraction.md

Several of the above steps are noted to have very long run times (e.g., 2 weeks to complete alignment), so I will not be replicating them in this repo. Instead, I will use the processed methylation calls to begin differential methylation analysis.

How to work in this repo

The repo is structured into three primary directories:

• code

• data

• output

All scripts, R code, and rendered .md and .html files should be stored in code. All input data (e.g., genomes) should be stored in data. All output files from running code should be stored in output. Note that files larger then 100 MB cannot be stored on Github.

In code, files will be organized with a numerical prefix. Each distinct task should be performed in its own code file with its own two-digit number. For example:

• 01-trimming.Rmd

• 02-alignment.Rmd

• 03-deduplication.Rmd

In output, the outputs from a given script should be stored in a subdirectory of the same name. For example, with the above scripts, we would have the following subdirectories in output:

• 01-trimming

• 02-alignment

• 03-deduplication

To maintain the document numbering in the ceasmallr repo, I’ll begin differential methylation analysis with the 06- prefix.