---
title: "03-Peve bismark"
author: Steven Roberts
date: "`r format(Sys.time(), '%d %B, %Y')`"  
output: 
  html_document:
    theme: readable
    highlight: zenburn
    toc: true
    toc_float: true
    number_sections: true
    code_folding: show
    code_download: true
---

```{r setup, include=FALSE}
library(knitr)
library(tidyverse)
knitr::opts_chunk$set(
  echo = TRUE,         # Display code chunks
  eval = FALSE,         # Evaluate code chunks
  warning = FALSE,     # Hide warnings
  message = FALSE,     # Hide messages
  fig.width = 6,       # Set plot width in inches
  fig.height = 4,      # Set plot height in inches
  fig.align = "center" # Align plots to the center
)
```



```{bash}
cd ../data
curl -O https://gannet.fish.washington.edu/seashell/snaps/Porites_evermanni_v1.fa
```




# ALignment

```{bash}
# Directories and programs
bismark_dir="/programs/Bismark-0.21.0"
bowtie2_dir="/home/shared/bowtie2-2.4.4-linux-x86_64"
genome_folder="../data/"

/home/shared/Bismark-0.24.0/bismark_genome_preparation \
--path_to_aligner ${bowtie2_dir} \
--verbose \
--parallel 28 \
${genome_folder}
```


https://gannet.fish.washington.edu/gitrepos/urol-e5/timeseries_molecular/E-Peve/output/01.00-E-Peve-WGBS-trimming-fastp-FastQC-MultiQC/

```{bash}
wget -r \
--no-directories --no-parent \
-P ../data/03-Peve-bismark \
-A "*gz" https://gannet.fish.washington.edu/gitrepos/urol-e5/timeseries_molecular/E-Peve/output/01.00-E-Peve-WGBS-trimming-fastp-FastQC-MultiQC/
```


```{bash}
reads_dir="../data/03-Peve-bismark/"
genome_folder="../data/"
output_dir="../output/03-Peve-bismark/"

mkdir -p "${output_dir}"

find ${reads_dir}*_R1_001.fastp-trim.fq.gz \
| xargs -n 1 basename -s _R1_001.fastp-trim.fq.gz \
| while read sample; do
    # Define the expected output file
    expected_output="${output_dir}${sample}_R1_001.fastp-trim_bismark_bt2_pe.bam"

    # Check if the expected output file exists before running
    if [[ ! -f "${expected_output}" ]]; then
        echo "Processing sample: ${sample}"
        /home/shared/Bismark-0.24.0/bismark \
            --genome ${genome_folder} \
            -p 8 \
            -u 10000 \
            --path_to_bowtie2 /home/shared/bowtie2-2.4.4-linux-x86_64 \
            --score_min L,0,-0.6 \
            -1 ${reads_dir}${sample}_R1_001.fastp-trim.fq.gz \
            -2 ${reads_dir}${sample}_R2_001.fastp-trim.fq.gz \
            -o "${output_dir}" \
            > "${output_dir}${sample}_bismark.out" 2> "${output_dir}${sample}_bismark.err"
    else
        echo "Skipping ${sample}, output already exists."
    fi
done
```









#dedup

```{bash}
find /gscratch/srlab/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/output/02.20-D-Apul-WGBS-alignment-SLURM_array-bismark/*.bam | \
xargs -n 1 basename -s _R1_001.fastp-trim_bismark_bt2_pe.bam | \
parallel -j 8 deduplicate_bismark \
--bam \
--paired \
--output_dir ../output/15-Apul-bismark \
/gscratch/srlab/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/output/02.20-D-Apul-WGBS-alignment-SLURM_array-bismark//{}_R1_001.fastp-trim_bismark_bt2_pe.bam
```








```{bash}
find ../output/15-Apul-bismark/*deduplicated.bam | \
xargs basename -s _R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bam | \
xargs -I{} samtools \
sort --threads 24 \
../output/15-Apul-bismark/{}_R1_001.fastp-trim_bismark_bt2_pe.deduplicated.bam \
-o ../output/15-Apul-bismark/{}.sorted.bam
```

# methylation exraction

```
find ../output/09-meth-quant/*deduplicated.bam | xargs -n 1 -I{} /home/shared/Bismark-0.24.0/bismark_methylation_extractor --bedGraph --counts --comprehensive --merge_non_CpG --multicore 24 --buffer_size 75% --output ../output/09-meth-quant {} 
```

```{bash}
find ../output/15-Apul-bismark/*deduplicated.bam | xargs -n 1 -I{} \
bismark_methylation_extractor --bedGraph --counts --comprehensive --merge_non_CpG \
--multicore 24 --buffer_size 75% --output ../output/15-Apul-bismark "{}"
```

# Methylation call

```
find ../output/09-meth-quant/*deduplicated.bismark.cov.gz | \
xargs -n 1 basename -s _pe.deduplicated.bismark.cov.gz | \
parallel -j 48 /home/shared/Bismark-0.24.0/coverage2cytosine \
--genome_folder ../data/ \
-o ../output/09-meth-quant/{} \
--merge_CpG \
--zero_based \
../output/09-meth-quant/{}_pe.deduplicated.bismark.cov.gz
```

```{bash}
find ../output/15-Apul-bismark/*deduplicated.bismark.cov.gz | \
xargs -n 1 basename -s _pe.deduplicated.bismark.cov.gz | \
parallel -j 24 coverage2cytosine \
--genome_folder /gscratch/srlab/sam/gitrepos/urol-e5/timeseries_molecular/D-Apul/data/ \
-o ../output/15-Apul-bismark/{} \
--merge_CpG \
--zero_based \
../output/15-Apul-bismark/{}_pe.deduplicated.bismark.cov.gz
```

```{bash}
head ../output/15-Apul-bismark/1H3*evidence.cov
```