01.00-trimming-fastp-fastqc
================
Sam White
2024-12-07

- <a href="#1-description" id="toc-1-description">1 Description</a>
  - <a href="#11-inputs" id="toc-11-inputs">1.1 Inputs:</a>
  - <a href="#12-outputs" id="toc-12-outputs">1.2 Outputs:</a>
- <a href="#2-create-a-bash-variables-file"
  id="toc-2-create-a-bash-variables-file">2 Create a Bash variables
  file</a>
- <a href="#3-create-adapters-fasta-for-use-with-fastp-trimming"
  id="toc-3-create-adapters-fasta-for-use-with-fastp-trimming">3 Create
  adapters FastA for use with <code>fastp</code> trimming</a>
- <a href="#4-trimming-and-merging-with-fastp"
  id="toc-4-trimming-and-merging-with-fastp">4 Trimming and merging with
  fastp</a>
- <a href="#5-fastqcmultiqc-on-trimmed-reads"
  id="toc-5-fastqcmultiqc-on-trimmed-reads">5 FastQC/MultiQC on trimmed
  reads</a>
- <a href="#6-list-output-files" id="toc-6-list-output-files">6 List
  output files</a>

# 1 Description

This notebook will trim and merge R1 and R2 reads. The max length of
31bp is based on the `fastp` insert peak size from previous trimming
tests based on the the adapter and polyG trimming results, and previous
evaluation of mean read lengths via
[`FastQC`](https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
and [`MultiQC`](https://multiqc.info/).

## 1.1 Inputs:

- sRNAseq paired-end FastQs (e.g. `*.fastq.gz`)

## 1.2 Outputs:

- `*.fastqc.html`: FastQC results, in HTML format.

- `*fastp-adapters-polyG-31bp-merged.fq.gz`: Trimmed and merged reads
  with final length of 31bp.

- `multiqc_report.html`: A summary report of the alignment results
  generated by [MultiQC](https://github.com/MultiQC/MultiQC), in HTML
  format.

Libraries were prepared and sequenced by Azenta:

- Library prep: [NEB nebnext-small-rna-library-prep-set-for-illumina
  kit](https://www.neb.com/en-us/-/media/nebus/files/manuals/manuale7300_e7330_e7560_e7580.pdf?rev=d0964a2e637843b1afcb9f7d666d07b2&hash=7AC0B0EB012708EFAB0E4DBEEAF1446A)
  (PDF)

- Sequencing: Illumina HiSeq 4000, 150bp PE

Due to large file sizes of FastQs, they cannot be added to GitHub. Full
output from this notebook are available here:

- <https://gannet.fish.washington.edu/gitrepos/project-clam-oa/output/01.00-trimming-fastp-fastqc>

# 2 Create a Bash variables file

This allows usage of Bash variables across R Markdown chunks.

``` bash
{
echo "#### Assign Variables ####"
echo ""

echo "# Data directories"
echo 'export repo_dir=/home/shared/8TB_HDD_01/sam/gitrepos/RobertsLab/project-clam-oa'
echo 'export output_dir_top=${repo_dir}/output/01.00-trimming-fastp-fastqc'
echo 'export raw_reads_dir="${repo_dir}/output/00.00-fastqc-concatenation-raw_reads"'
echo 'export trimmed_fastqs_dir="${output_dir_top}"'
echo ""

echo "# Paths to programs"
echo 'export programs_dir="/home/shared"'
echo 'export fastp="${programs_dir}/fastp-v0.24.0/fastp"'
echo 'export fastqc="${programs_dir}/FastQC-0.12.1/fastqc"'
echo 'export multiqc="/home/sam/programs/mambaforge/bin/multiqc"'
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 "export trimmed_fastq_pattern='*fastp-trim*.fq.gz'"
echo ""

echo "# Input/output files"
echo 'export fastq_checksums=input_fastq_checksums.md5'
echo 'export NEB_adapters_fasta=NEB-adapters.fasta'
echo ""

echo "## NEB nebnext-small-rna-library-prep-set-for-illumina adapters"
echo 'export first_adapter="AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC"'
echo 'export second_adapter="GATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGCCGTATCATT"'
echo ""

echo "# Set maximum read length for fastp merging"
echo 'export max_read_length="31"'
echo ""

echo "# Set number of CPUs to use"
echo 'export threads=40'
echo ""


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

echo "# Print formatting"
echo 'export line="--------------------------------------------------------"'
echo ""
} > .bashvars

cat .bashvars
```

    #### Assign Variables ####

    # Data directories
    export repo_dir=/home/shared/8TB_HDD_01/sam/gitrepos/RobertsLab/project-clam-oa
    export output_dir_top=${repo_dir}/output/01.00-trimming-fastp-fastqc
    export raw_reads_dir="${repo_dir}/output/00.00-fastqc-concatenation-raw_reads"
    export trimmed_fastqs_dir="${output_dir_top}"

    # Paths to programs
    export programs_dir="/home/shared"
    export fastp="${programs_dir}/fastp-v0.24.0/fastp"
    export fastqc="${programs_dir}/FastQC-0.12.1/fastqc"
    export multiqc="/home/sam/programs/mambaforge/bin/multiqc"

    # Set FastQ filename patterns
    export fastq_pattern='*.fastq.gz'
    export R1_fastq_pattern='*_R1_*.fastq.gz'
    export R2_fastq_pattern='*_R2_*.fastq.gz'
    export trimmed_fastq_pattern='*fastp-trim*.fq.gz'

    # Input/output files
    export fastq_checksums=input_fastq_checksums.md5
    export NEB_adapters_fasta=NEB-adapters.fasta

    ## NEB nebnext-small-rna-library-prep-set-for-illumina adapters
    export first_adapter="AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC"
    export second_adapter="GATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGCCGTATCATT"

    # Set maximum read length for fastp merging
    export max_read_length="31"

    # Set number of CPUs to use
    export threads=40

    ## Inititalize arrays
    export fastq_array_R1=()
    export fastq_array_R2=()
    export trimmed_fastqs_array=()
    export R1_names_array=()
    export R2_names_array=()

    # Print formatting
    export line="--------------------------------------------------------"

# 3 Create adapters FastA for use with [`fastp`](https://github.com/OpenGene/fastp) trimming

``` bash
# Load bash variables into memory
source .bashvars

# Create output directory, if it doesn't exist
mkdir --parents "${output_dir_top}"

echo "Creating adapters FastA."
echo ""
adapter_count=0

# Check for adapters file first
# Then create adapters file if doesn't exist
if [ -f "${output_dir_top}/${NEB_adapters_fasta}" ]; then
  echo "${output_dir_top}/${NEB_adapters_fasta} already exists. Nothing to do."
else
  for adapter in "${first_adapter}" "${second_adapter}"
  do
    adapter_count=$((adapter_count + 1))
    printf ">%s\n%s\n" "adapter_${adapter_count}" "${adapter}"
  done >> "${output_dir_top}/${NEB_adapters_fasta}"
fi

echo ""
echo "Adapters FastA:"
echo ""
cat "${output_dir_top}/${NEB_adapters_fasta}"
echo ""
```

    Creating adapters FastA.


    Adapters FastA:

    >adapter_1
    AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC
    >adapter_2
    GATCGTCGGACTGTAGAACTCTGAACGTGTAGATCTCGGTGGTCGCCGTATCATT

# 4 Trimming and merging with fastp

``` bash
# Load bash variables into memory
source .bashvars

# Create output directory, if it doesn't exist.
mkdir --parents "${trimmed_fastqs_dir}"

# Change to directory with raw reads
cd "${raw_reads_dir}"

# Create arrays of FastQ R1 files and sample names
# Do NOT quote R1_fastq_pattern variable
for fastq in ${R1_fastq_pattern}
do
  fastq_array_R1+=("${fastq}")

  # Use parameter substitution to remove all text up to and including last "." from
  # right side of string.
  R1_names_array+=("${fastq%%.*}")
done

# Create array of FastQ R2 files
# Do NOT quote R2_fastq_pattern variable
for fastq in ${R2_fastq_pattern}
do
  fastq_array_R2+=("${fastq}")

  # Use parameter substitution to remove all text up to and including last "." from
  # right side of string.
  R2_names_array+=("${fastq%%.*}")
done

############ RUN FASTP ############
# Uses parameter substitution (e.g. ${R1_sample_name%%_*})to rm the _R[12]
# Uses NEB adapter file


# Run fastp on files
echo "Beginning fastp trimming."
echo ""

time \
for index in "${!fastq_array_R1[@]}"
do
  # Get sample name
  R1_sample_name="${R1_names_array[index]%%_*}"
  R2_sample_name="${R2_names_array[index]%%_*}"

  # Save merged sample name
  merged_sample_name="${R1_sample_name}-fastp-adapters-polyG-${max_read_length}bp-merged"
  
  # Begin fastp trimming
  ${fastp} \
  --in1 ${fastq_array_R1[index]} \
  --in2 ${fastq_array_R2[index]} \
  --adapter_fasta ${output_dir_top}/${NEB_adapters_fasta} \
  --trim_poly_g \
  --overlap_len_require 17 \
  --length_limit ${max_read_length} \
  --merge \
  --merged_out ${trimmed_fastqs_dir}/${merged_sample_name}.fq.gz \
  --thread ${threads} \
  --html "${trimmed_fastqs_dir}/${merged_sample_name}.html" \
  --json "${trimmed_fastqs_dir}/${merged_sample_name}.json" \
  --report_title "${trimmed_fastqs_dir}/${merged_sample_name}" \
  2> ${trimmed_fastqs_dir}/${merged_sample_name}.stderr
    
  # Move to trimmed directory
  # This is done so checksums file doesn't include excess path
  cd ${trimmed_fastqs_dir}

  # Generate md5 checksums for newly trimmed files
  md5sum "${merged_sample_name}.fq.gz" | tee --append "${merged_sample_name}.fq.gz.md5"
    
  # Change back to to raw reads directory
  cd "${raw_reads_dir}"

done

echo ""
echo "fastp trimming complete."
echo ""

############ END fastp ############
```

    Beginning fastp trimming.

    51f055f1810a19deb2ca09ea5bb9df0b  196-fastp-adapters-polyG-31bp-merged.fq.gz
    a0673f4ad1fda1fe3f375aa2c76d5fa3  199-fastp-adapters-polyG-31bp-merged.fq.gz
    b989841536ec95b3aeb10ef20f848ea7  211-fastp-adapters-polyG-31bp-merged.fq.gz
    c33d44ac03a2095e55c3167b35fde0da  24-fastp-adapters-polyG-31bp-merged.fq.gz
    02ddae1e57b7b9e7e63d74b4e1f725b8  260-fastp-adapters-polyG-31bp-merged.fq.gz
    7c54b3c782c4ed0f15625243dfacc10a  26-fastp-adapters-polyG-31bp-merged.fq.gz
    ca7286f6967abf299a9557f204eee659  30-fastp-adapters-polyG-31bp-merged.fq.gz
    96c1e31ce89b5caa8a59b54c2745ae1e  310-fastp-adapters-polyG-31bp-merged.fq.gz
    6d5a35136a20d9da975cf8740493a711  33-fastp-adapters-polyG-31bp-merged.fq.gz
    b9c94cba5c2d2df557a833ab3a868763  341-fastp-adapters-polyG-31bp-merged.fq.gz
    7530a8c684b78aa86dee725a71601f8e  34-fastp-adapters-polyG-31bp-merged.fq.gz
    7c898a66f9119084ca290f0d85a34f12  35-fastp-adapters-polyG-31bp-merged.fq.gz
    d80f6b589e5228731de750dd6ec5dac9  363-fastp-adapters-polyG-31bp-merged.fq.gz
    f77f9aa54e2c6662abe24c3a063b6006  367-fastp-adapters-polyG-31bp-merged.fq.gz
    a2281f8237ae8cb385a2c22bd185fe57  376-fastp-adapters-polyG-31bp-merged.fq.gz
    03b221d3f58f3efb6a6c9a7dcfd37233  460-fastp-adapters-polyG-31bp-merged.fq.gz
    c266d11555b76213d739626d40715dde  485-fastp-adapters-polyG-31bp-merged.fq.gz
    3fdd30605d7a94c36b9191b34e06fbd3  501-fastp-adapters-polyG-31bp-merged.fq.gz
    6929563775fded5a7a9023e3b87d76cd  71-fastp-adapters-polyG-31bp-merged.fq.gz
    048d7334f58f5ce149aeadc5745fc28a  88-fastp-adapters-polyG-31bp-merged.fq.gz

    real    11m50.402s
    user    140m1.165s
    sys 3m20.369s

    fastp trimming complete.

# 5 FastQC/MultiQC on trimmed reads

``` bash
# Load bash variables into memory
source .bashvars

# Create output directory, if it doesn't exist.
mkdir --parents "${trimmed_fastqs_dir}"

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

### NOTE: Do NOT quote raw_fastqc_list
# Create array of trimmed FastQs
trimmed_fastqs_array=(${trimmed_fastqs_dir}/*merged.fq.gz)

# 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
${fastqc} \
--threads ${threads} \
--outdir ${trimmed_fastqs_dir} \
--quiet \
${trimmed_fastqc_list}

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

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

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

${multiqc} ${trimmed_fastqs_dir} -o ${trimmed_fastqs_dir}

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

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

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

    Beginning FastQC on raw reads...

    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    application/gzip
    FastQC on trimmed reads complete!

    Beginning MultiQC on raw FastQC...


      /// MultiQC 🔍 | v1.14

    |           multiqc | MultiQC Version v1.25.2 now available!
    |           multiqc | Search path : /home/shared/8TB_HDD_01/sam/gitrepos/RobertsLab/project-clam-oa/output/01.00-trimming-fastp-fastqc
    |         searching | ━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━ 100% 141/141  
    |            fastqc | Found 20 reports
    |           multiqc | Compressing plot data
    |           multiqc | Report      : ../output/01.00-trimming-fastp-fastqc/multiqc_report.html
    |           multiqc | Data        : ../output/01.00-trimming-fastp-fastqc/multiqc_data
    |           multiqc | MultiQC complete

    MultiQC on trimmed FastQs complete.

    Removing FastQC zip files.

    FastQC zip files removed.

# 6 List output files

``` bash
# Load bash variables into memory
source .bashvars

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

    total 6.8G
    -rw-r--r-- 1 sam sam 564K Dec  5 18:48 196-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 336M Dec  5 18:36 196-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:36 196-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:36 196-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:36 196-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:36 196-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 569K Dec  5 18:48 199-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 283M Dec  5 18:37 199-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:37 199-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:37 199-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:37 199-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:37 199-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 567K Dec  5 18:48 211-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 298M Dec  5 18:37 211-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:37 211-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 349K Dec  5 18:37 211-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  91K Dec  5 18:37 211-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:37 211-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 566K Dec  5 18:48 24-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 320M Dec  5 18:38 24-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   76 Dec  5 18:38 24-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:38 24-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:38 24-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.2K Dec  5 18:38 24-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 569K Dec  5 18:48 260-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 327M Dec  5 18:38 260-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:38 260-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:38 260-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:38 260-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:38 260-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 568K Dec  5 18:48 26-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 306M Dec  5 18:39 26-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   76 Dec  5 18:39 26-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 349K Dec  5 18:39 26-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  91K Dec  5 18:39 26-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.2K Dec  5 18:39 26-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 570K Dec  5 18:48 30-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 331M Dec  5 18:39 30-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   76 Dec  5 18:39 30-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:39 30-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:39 30-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.2K Dec  5 18:39 30-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 571K Dec  5 18:48 310-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 326M Dec  5 18:40 310-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:40 310-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:40 310-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:40 310-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:40 310-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 566K Dec  5 18:49 33-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 341M Dec  5 18:41 33-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   76 Dec  5 18:41 33-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 351K Dec  5 18:40 33-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:40 33-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.2K Dec  5 18:41 33-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 564K Dec  5 18:48 341-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 325M Dec  5 18:41 341-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:41 341-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:41 341-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:41 341-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:41 341-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 564K Dec  5 18:49 34-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 394M Dec  5 18:42 34-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   76 Dec  5 18:42 34-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:42 34-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:42 34-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.2K Dec  5 18:42 34-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 570K Dec  5 18:48 35-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 298M Dec  5 18:42 35-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   76 Dec  5 18:42 35-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 349K Dec  5 18:42 35-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  91K Dec  5 18:42 35-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.2K Dec  5 18:42 35-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 572K Dec  5 18:49 363-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 354M Dec  5 18:43 363-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:43 363-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:43 363-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:43 363-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:43 363-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 564K Dec  5 18:49 367-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 323M Dec  5 18:43 367-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:43 367-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 349K Dec  5 18:43 367-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  91K Dec  5 18:43 367-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:43 367-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 566K Dec  5 18:49 376-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 603M Dec  5 18:44 376-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:44 376-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 351K Dec  5 18:44 376-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  93K Dec  5 18:44 376-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:44 376-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 565K Dec  5 18:48 460-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 305M Dec  5 18:45 460-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:45 460-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:45 460-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:45 460-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:45 460-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 566K Dec  5 18:49 485-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 320M Dec  5 18:46 485-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:46 485-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:46 485-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:46 485-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:46 485-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 563K Dec  5 18:49 501-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 353M Dec  5 18:46 501-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   77 Dec  5 18:46 501-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:46 501-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:46 501-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.3K Dec  5 18:46 501-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 568K Dec  5 18:49 71-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 380M Dec  5 18:47 71-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   76 Dec  5 18:47 71-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:47 71-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:47 71-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.2K Dec  5 18:47 71-fastp-adapters-polyG-31bp-merged.stderr
    -rw-r--r-- 1 sam sam 563K Dec  5 18:49 88-fastp-adapters-polyG-31bp-merged_fastqc.html
    -rw-r--r-- 1 sam sam 374M Dec  5 18:47 88-fastp-adapters-polyG-31bp-merged.fq.gz
    -rw-r--r-- 1 sam sam   76 Dec  5 18:47 88-fastp-adapters-polyG-31bp-merged.fq.gz.md5
    -rw-r--r-- 1 sam sam 350K Dec  5 18:47 88-fastp-adapters-polyG-31bp-merged.html
    -rw-r--r-- 1 sam sam  92K Dec  5 18:47 88-fastp-adapters-polyG-31bp-merged.json
    -rw-r--r-- 1 sam sam 2.2K Dec  5 18:47 88-fastp-adapters-polyG-31bp-merged.stderr
    drwxr-xr-x 2 sam sam 4.0K Dec  5 18:49 multiqc_data
    -rw-r--r-- 1 sam sam 1.3M Dec  5 18:49 multiqc_report.html
    -rw-r--r-- 1 sam sam  113 Dec  5 18:36 NEB-adapters.fasta