### Background 

This code will align trimmed _P.tuahiniensis_ EM-seq data to the _P.tuahiniensis_ genome using Bismark and create loci counts matrices using the [nf-core Methylseq pipeline](https://nf-co.re/methylseq/3.0.0/).


**Input data:**

- [Trimmed fastqs](https://gannet.fish.washington.edu/gitrepos/urol-e5/timeseries_molecular/F-Ptua/output/01.00-F-Ptua-WGBS-trimming-fastp-FastQC-MultiQC/)
- [genome](https://owl.fish.washington.edu/halfshell/genomic-databank/Pocillopora_meandrina_HIv1.assembly.fasta)


**Files needed to run nextflow pipeline:**

- [config file](https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/uw_hyak_srlab.config)
- [samplesheet](https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/samplesheet.csv)

### Methods
**Copy genome files to klone**

```
# show path
pwd
/gscratch/srlab/strigg/GENOMES

# copy genome
wget https://owl.fish.washington.edu/halfshell/genomic-databank/Pocillopora_meandrina_HIv1.assembly.fasta

```


**Copy EM-seq data to Klone**

```
# open screen session (reopened existing session)
screen -S methylseq

# start interactive node
salloc -A srlab -p cpu-g2-mem2x -N 1 -c 1 --mem=16GB --time=16:00:00

# copy data
rsync --progress --verbose --archive shellytrigg@gannet.fish.washington.edu:/volume2/web/gitrepos/urol-e5/timeseries_molecular/F-Ptua/output/01.00-F-Ptua-WGBS-trimming-fastp-FastQC-MultiQC/*.gz /gscratch/scrubbed/strigg/analyses/20250421_methylseq
```

**Run nf-core Methylseq pipeline on Klone**

```
# open screen session 
screen -S methylseq

# start interactive node
salloc -A srlab -p cpu-g2-mem2x -N 1 -c 1 --mem=16GB --time=72:00:00

# activate conda environment. Note: you can use `conda` instead of `mamba` 
mamba activate /gscratch/srlab/nextflow/bin/miniforge/envs/nextflow

# run pipeline
nextflow run nf-core/methylseq \
-c /gscratch/srlab/strigg/bin/uw_hyak_srlab.config \
--input /gscratch/scrubbed/strigg/analyses/20250422_methylseq/samplesheet.csv \
--outdir /gscratch/scrubbed/strigg/analyses/20250422_methylseq \
--fasta /gscratch/srlab/strigg/GENOMES/Pocillopora_meandrina_HIv1.assembly.fasta \
--em_seq \
-resume \
-with-report nf_report.html \
-with-trace \
-with-timeline nf_timeline.html \
--skip_trimming \
--nomeseq 

```

### Results

- Multiqc report: [https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/multiqc/bismark/multiqc_report.html](https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/multiqc/bismark/multiqc_report.html)
- Bismark summary report: [https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/bismark/summary/bismark_summary_report.html](https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/bismark/summary/bismark_summary_report.html)
- Pipeline report: [https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/nf_report.html](https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/nf_report.html)
- Pipeline timeline: [https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/nf_timeline.html](https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/nf_timeline.html)
- Counts matrices: [https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/bismark/methylation_calls/methylation_coverage/](https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/bismark/methylation_calls/methylation_coverage/)
	- <sample_name>.fastp-trim_bismark_bt2_pe.deduplicated.bismark.cov.gz
- Deduplicated sorted bam files: [https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/bismark/deduplicated/](https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/bismark/deduplicated/) 
	- <sample_name>.deduplicated.sorted.bam 
- Other bismark output: [https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/bismark/](https://gannet.fish.washington.edu/metacarcinus/E5/Ptuahiniensis/20250422_methylseq/bismark/)