Project name: PSMFC-mytilus-byssus-pilot
Funding source: Pacific States Marine Fisheries Commission
Species: mytilus galloprovincialis, mytilus trossulus
variable: OA, DO, seawater temperature, desiccation

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Download and QC tag-seq data received on 7/14/2022

All following code chunks were run on Raven

Download tag-seq data

mkdir raw-data/
cd raw-data/

wget -r \
--no-directories --no-parent \
-P . \
-A .fastq.gz https://gannet.fish.washington.edu/panopea/PSMFC-mytilus-byssus-pilot/20220405-tagseq/ \
--no-check-certificate

unzip .fastq.gz files

cd raw-data/
gunzip *.fastq.gz

Run fastqc on untrimmed files

mkdir fastqc/
mkdir fastqc/untrimmed/

/home/shared/FastQC/fastqc \
/home/shared/8TB_HDD_02/mattgeorgephd/PSMFC-mytilus-byssus-pilot/raw-data/*.fastq \
--outdir /home/shared/8TB_HDD_02/mattgeorgephd/PSMFC-mytilus-byssus-pilot/fastqc/untrimmed/ \
--quiet

Run multiqc

eval "$(/opt/anaconda/anaconda3/bin/conda shell.bash hook)"
conda activate

cd fastqc/untrimmed/

multiqc .

Link to multiQC report untrimmed sequences

trim adapter sequences

mkdir trim-fastq/
cd raw-data

for F in *.fastq
do
#strip .fastq and directory structure from each file, then
# add suffice .trim to create output name for each file
results_file="$(basename -a $F)"

# run cutadapt on each file
/home/shared/8TB_HDD_02/mattgeorgephd/.local/bin/cutadapt $F -a A{8} -a G{8} -a AGATCGG -u 15 -m 20 -o \
/home/shared/8TB_HDD_02/mattgeorgephd/PSMFC-mytilus-byssus-pilot/trim-fastq/$results_file
done

Run fastqc on trimmed files

mkdir fastqc/
mkdir fastqc/trimmed/

/home/shared/FastQC/fastqc \
/home/shared/8TB_HDD_02/mattgeorgephd/PSMFC-mytilus-byssus-pilot/merged-fastq/*.fastq \
--outdir /home/shared/8TB_HDD_02/mattgeorgephd/PSMFC-mytilus-byssus-pilot/fastqc/trimmed/ \
--quiet

Run multiqc on trimmed files

eval "$(/opt/anaconda/anaconda3/bin/conda shell.bash hook)"
conda activate

cd fastqc/trimmed/

multiqc .

concatenate fastq files by lane

mkdir merged-fastq
cd trim-fastq/

printf '%s\n' *.fastq | sed 's/^\([^_]*_[^_]*\).*/\1/' | uniq |
while read prefix; do
    cat "$prefix"*R1*.fastq >"${prefix}_R1.fastq"
    # cat "$prefix"*R2*.fastq >"${prefix}_R2.fastq" # include if more than one run
done

# I moved files to merged-fastq

Link to multiQC report on trimmed and concatenated sequences

Generate de novo transcriptome from available RNA-seq datasets

[RAVEN] Download mytilus trossulus transcriptome - https://www.ncbi.nlm.nih.gov/sra/SRX3198554[accn]

Tutorial: https://blogs.iu.edu/ncgas/2021/02/22/a-beginners-guide-to-the-sra/

# /home/shared/sratoolkit.2.11.2-ubuntu64/bin/vdb-config --interactive

mkdir SRA/
cd SRA/

# Download SRA accession list - all 47 mytilus trossulus
wget -r \
--no-directories --no-parent \
-P . \
-A SraAccList.txt https://gannet.fish.washington.edu/panopea/PSMFC-mytilus-byssus-pilot/genome/ \
--no-check-certificate

# Download SRA files using SRA accession list
/home/shared/sratoolkit.2.11.2-ubuntu64/bin/prefetch \
--option-file SraAccList.txt

# Move .sra files out of containing folders to SRA
mv */* .

# convert files to fastq
for file in *.sra; do /home/shared/sratoolkit.2.11.2-ubuntu64/bin/fasterq-dump "${file}"; done # e -48

Transfer SRA files to Gannet

rsync --archive --progress --verbose --relative ./SRA mngeorge@gannet.fish.washington.edu:/volume2/web/panopea/PSMFC-mytilus-byssus-pilot

Running Trinity on Mox

Login to mox

ssh mngeorge@mox.hyak.uw.edu

Login to Gannet and copy fastq files to gscratch

ssh mngeorge@gannet.fish.washington.edu <login>
cd /volume2/web/panopea/PSMFC-mytilus-byssus-pilot/
rsync --archive --progress --verbose --relative ./20220405-tagseq mngeorge@mox.hyak.uw.edu:/gscratch/srlab/mngeorge/data/mtrossulus

rsync --archive --progress --verbose --relative ./SRA mngeorge@mox.hyak.uw.edu:/gscratch/srlab/mngeorge/data/mtrossulus

#the serve was disconnected half way through, so I ran this:

rsync --archive --progress --verbose --relative --ignore-existing --dry-run ./sbatch_scripts mngeorge@mox.hyak.uw.edu:/gscratch/srlab/mngeorge/sbatch_scripts

Add paths to ~/.bashrc file

# Open bashrc w/ nano
nano ~/.bashrc




# Check inclusion
echo "$PATH//:/$'\n'}"

Add job to queue

# Added 9/16/2022 - data on scrubbed for only 21 days
cd /gscratch/scrubbed/mngeorge/sbatch_scripts
sbatch 202208_PSMFC-mytilus-byssus-pilot_trinity.sh

Check Job Status

scontrol show job 3897217

or

squeue