---
title: "01-Apul-RNA-trimming-FastQC"
author: "Kathleen Durkin"
date: "2024-08-19"
always_allow_html: true
output: 
  bookdown::html_document2:
    theme: cosmo
    toc: true
    toc_float: true
    number_sections: true
    code_folding: show
    code_download: true
  github_document:
    toc: true
    toc_depth: 3
    number_sections: true
    html_preview: true 
---

```{r setup, include=FALSE}
library(knitr)
knitr::opts_chunk$set(
  echo = TRUE,         # Display code chunks
  eval = FALSE,        # Evaluate code chunks
  warning = FALSE,     # Hide warnings
  message = FALSE,     # Hide messages
  comment = ""         # Prevents appending '##' to beginning of lines in code output
)
```

Code for trimming and QCing RNAseq data, to be used on *Acropora pulchra*.


For now I'm just going to QC the [raw reads](https://owl.fish.washington.edu/nightingales/A_pulchra/30-789513166/) and the [trimmed reads](https://gannet.fish.washington.edu/Atumefaciens/20230519-E5_coral-fastqc-fastp-multiqc-RNAseq/A_pulchra/trimmed/) generated as a part of the [deep-dive](https://github.com/urol-e5/deep-dive/tree/main) project. If additional/different trimming needs to be done for this expression work, it will be performed here.


Inputs:

- RNA-seq gzipped FastQs (e.g. `*.fastq.gz`)

Outputs:

- [`FastQC`](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) HTML reports for raw and trimmed reads.

- [`MultiQC`](https://multiqc.info/) HTML summaries of [`FastQC`](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for raw and trimmed reads.


[Sequencing report](https://owl.fish.washington.edu/nightingales/P_evermanni/30-789513166/Azenta_30-789513166_Data_Report.html)
[Trimming details](https://robertslab.github.io/sams-notebook/posts/2023/2023-05-19-FastQ-QC-and-Trimming---E5-Coral-RNA-seq-Data-for-A.pulchra-P.evermanni-and-P.meandrina-Using-FastQC-fastp-and-MultiQC-on-Mox/)

---

# Create a Bash variables file

This allows usage of Bash variables (e.g. paths to common directories) across R Markdown chunks.
```{r save-bash-variables-to-rvars-file, engine='bash', eval=TRUE}
{
echo "#### Assign Variables ####"
echo ""

echo "# Data directories"
echo 'export expression_dir=/home/shared/8TB_HDD_02/shedurkin/deep-dive-expression'
echo 'export output_dir_top=${expression_dir}/D-Apul/output/01-Apul-RNA-trimming-FastQC'
echo 'export raw_fastqc_dir=${output_dir_top}/raw-fastqc'
echo 'export raw_reads_dir=${expression_dir}/D-Apul/data/01-Apul-RNA-trimming-FastQC/raw-reads'
echo 'export raw_reads_url="https://owl.fish.washington.edu/nightingales/A_pulchra/30-789513166/"'
echo 'export trimmed_fastqc_dir=${output_dir_top}/trimmed-fastqc'
echo 'export trimmed_reads_dir=${expression_dir}/D-Apul/data/01-Apul-RNA-trimming-FastQC/trimmed-reads'
echo 'export trimmed_reads_url="https://gannet.fish.washington.edu/Atumefaciens/20230519-E5_coral-fastqc-fastp-multiqc-RNAseq/A_pulchra/trimmed/"'
echo ""

echo "# Paths to programs"
echo 'export fastqc=/home/shared/FastQC-0.12.1/fastqc'
echo 'export multiqc=/home/sam/programs/mambaforge/bin/multiqc'
echo 'export flexbar=/home/shared/flexbar-3.5.0-linux/flexbar'
echo ""

echo "# Set FastQ filename patterns"
echo "export fastq_pattern='*.fastq.gz'"
echo "export R1_fastq_pattern='*_R1_*.fastq.gz'"
echo "export R2_fastq_pattern='*_R2_*.fastq.gz'"
echo ""

echo "# Set number of CPUs to use"
echo 'export threads=20'
echo ""

echo "# Input/output files"
echo 'export raw_checksums=checksums.md5'
echo 'export trimmed_checksums=trimmed_fastq_checksums.md5'
echo ""



echo "## Inititalize arrays"
echo 'export fastq_array_R1=()'
echo 'export fastq_array_R2=()'
echo 'export raw_fastqs_array=()'
echo 'export R1_names_array=()'
echo 'export R2_names_array=()'
echo 'export trimmed_fastqs_array=()'
echo ""

echo "# Programs associative array"
echo "declare -A programs_array"
echo "programs_array=("
echo '[fastqc]="${fastqc}" \'
echo '[multiqc]="${multiqc}" \'
echo '[flexbar]="${flexbar}"'
echo ")"
} > .bashvars

cat .bashvars
```


# Raw reads
## Download raw RNA-seq reads

Reads are downloaded from: https://owl.fish.washington.edu/nightingales/A_pulchra/30-789513166/

The `--cut-dirs 3` command cuts the preceding directory structure (i.e. `nightingales/A_pulchra/30-789513166/`) so that we just end up with 
the reads.

```{r download-raw-reads, engine='bash'}
# Load bash variables into memory
source .bashvars

wget \
--directory-prefix ${raw_reads_dir} \
--recursive \
--no-check-certificate \
--continue \
--cut-dirs 3 \
--no-host-directories \
--no-parent \
--quiet \
--accept ${fastq_pattern} ${raw_reads_url}

source .bashvars
```

```{r check-raw-reads, engine='bash', eval=TRUE}
# Load bash variables into memory
source .bashvars

ls -lh "${raw_reads_dir}"
```

## Verify raw read checksums
```{r verify-raw-read-checksums, engine='bash'}
# Load bash variables into memory
source .bashvars

wget \
--directory-prefix ${raw_reads_dir} \
--recursive \
--no-check-certificate \
--continue \
--cut-dirs 3 \
--no-host-directories \
--no-parent \
--quiet \
--accept checksums.md5 ${raw_reads_url}

cd "${raw_reads_dir}"

md5sum checksums.md5 --check
```


## FastQC/MultiQC on raw reads
```{r raw-fastqc-multiqc, engine='bash'}
# Load bash variables into memory
source .bashvars

############ RUN FASTQC ############


# Create array of raw FastQs
raw_fastqs_array=(${raw_reads_dir}/${fastq_pattern})

# Pass array contents to new variable as space-delimited list
raw_fastqc_list=$(echo "${raw_fastqs_array[*]}")

echo "Beginning FastQC on raw reads..."
echo ""

# Run FastQC
### NOTE: Do NOT quote raw_fastqc_list
${programs_array[fastqc]} \
--threads ${threads} \
--outdir ${raw_fastqc_dir} \
--quiet \
${raw_fastqc_list}

echo "FastQC on raw reads complete!"
echo ""

############ END FASTQC ############

############ RUN MULTIQC ############
echo "Beginning MultiQC on raw FastQC..."
echo ""

${programs_array[multiqc]} ${raw_fastqc_dir} -o ${raw_fastqc_dir}

echo ""
echo "MultiQC on raw FastQs complete."
echo ""

############ END MULTIQC ############

echo "Removing FastQC zip files."
echo ""
rm ${raw_fastqc_dir}/*.zip
echo "FastQC zip files removed."
echo ""
```

```{r check-raw-reads-QC-files, engine='bash', eval=TRUE}
# Load bash variables into memory
source .bashvars

# View directory contents
ls -lh ${raw_fastqc_dir}
```


# Trimmed reads
## Download trimmed RNA-seq reads

Reads are downloaded from: https://gannet.fish.washington.edu/Atumefaciens/20230519-E5_coral-fastqc-fastp-multiqc-RNAseq/A_pulchra/trimmed/

```{r download-trimmed-reads, engine='bash'}
# Load bash variables into memory
source .bashvars

wget \
--directory-prefix ${trimmed_reads_dir} \
--recursive \
--no-check-certificate \
--continue \
--cut-dirs 4 \
--no-host-directories \
--no-parent \
--quiet \
--accept ${fastq_pattern} ${trimmed_reads_url}
```

```{r check-trimmed-reads, engine='bash', eval=TRUE}
# Load bash variables into memory
source .bashvars

ls -lh "${trimmed_reads_dir}"
```

## Verify raw read checksums
```{r verify-trimmed-read-checksums, engine='bash'}
# Load bash variables into memory
source .bashvars

wget \
--directory-prefix ${trimmed_reads_dir} \
--recursive \
--no-check-certificate \
--continue \
--cut-dirs 4 \
--no-host-directories \
--no-parent \
--quiet \
--accept trimmed_fastq_checksums.md5 ${trimmed_reads_url}

cd "${trimmed_reads_dir}"

md5sum trimmed_fastq_checksums.md5 --check
```


## FastQC/MultiQC on trimmed reads

```{r FastQC-MultiQC-trimmed-reads, engine='bash'}
# Load bash variables into memory
source .bashvars

############ RUN FASTQC ############

### NOTE: Do NOT quote raw_fastqc_list
# Create array of trimmed FastQs
trimmed_fastqs_array=(${trimmed_reads_dir}/${fastq_pattern})

# Pass array contents to new variable as space-delimited list
trimmed_fastqc_list=$(echo "${trimmed_fastqs_array[*]}")

echo "Beginning FastQC on raw reads..."
echo ""

# Run FastQC
${programs_array[fastqc]} \
--threads ${threads} \
--outdir ${trimmed_fastqc_dir} \
--quiet \
${trimmed_fastqc_list}

echo "FastQC on trimmed reads complete!"
echo ""

############ END FASTQC ############

############ RUN MULTIQC ############
echo "Beginning MultiQC on raw FastQC..."
echo ""

${programs_array[multiqc]} ${trimmed_fastqc_dir} -o ${trimmed_fastqc_dir}

echo ""
echo "MultiQC on trimmed FastQs complete."
echo ""

############ END MULTIQC ############

echo "Removing FastQC zip files."
echo ""
rm ${trimmed_fastqc_dir}/*.zip
echo "FastQC zip files removed."
echo ""
```

```{r view-trimmed-reads-QC-files, engine='bash', eval=TRUE}
# Load bash variables into memory
source .bashvars

# View directory contents
ls -lh ${trimmed_fastqc_dir}
```

# Summary