Trimming - Yellow Island eDNA FastQs Using Fastp FastQC and MultiQC

2025
eDNA
Yellow Island
fastp
trimming
FastQC
MultiQC
project-eDNA-yellow
Author

Sam White

Published

April 18, 2025

INTRO

In preparation for QIIME 2 analysis for project-eDNA-yellow (GitHub repo) project, we needed to perform adapter/quality trimming of the eDNA FastQs. Additionally, the trimmed filenames were adjusted to account for downstream filename requirements of QIIME 2.

Note

The contents below are from markdown knitted from 01.00-trimming-fastp-fastqc-multiqc.Rmd (commit 9d2b4a6).

1 BACKGROUND

This Rmd file trims FastQ files using fastp (Chen 2023), followed by quality checks with FastQC and MultiQC (Ewels et al. 2016). Additionally, it renames the trimmed FastQ files to be compliant with QIIME 2 naming expectations.

Note

If you need to download the raw sequencing reads, please see 00.00-fastqc-multiqc-raw-reads.Rmd

Inputs:

  • Paired-end FastQs

    • Example filename format: Yell_July18_Site1f_S92_L001_R[12]_001.fastq.gz

Outputs:

  • Trimmed FastQs.

    • Example filename format: Yell_July18_Site1f_S92_L001_R1.fastp-trim.fastq.gz

  • fastp JSON files.

  • FastQC HTML files.

  • MultiQC HTML report.

Software requirements:

2 Set R variables

# Data directories
data_dir <- "/home/sam/gitrepos/RobertsLab/project-eDNA-yellow/data/"
raw_reads_dir <- "/home/sam/gitrepos/RobertsLab/project-eDNA-yellow/data/raw-fastqs"
output_dir <- "/home/sam/gitrepos/RobertsLab/project-eDNA-yellow/output/01.00-trimming-fastp-fastqc-multiqc"

# PROGRAMS
fastp <- "/home/sam/programs/fastp"
fastqc <- "/home/sam/programs/FastQC-0.12.1/fastqc"
multiqc <- "/home/sam/programs/miniforge3/bin/multiqc"

# CPUs
threads <- 4

# Define file patterns
R1_fastq_pattern <- "*_R1_001.fastq.gz"
R2_fastq_pattern <- "*_R2_001.fastq.gz"

# Export these as environment variables for bash chunks.
Sys.setenv(
  fastp = fastp,
  fastqc = fastqc,
  data_dir = data_dir,
  multiqc = multiqc,
  raw_reads_dir = raw_reads_dir,
  threads = threads,
  R1_fastq_pattern = R1_fastq_pattern,
  R2_fastq_pattern = R2_fastq_pattern,
  output_dir = output_dir
)

3 Fastp Trimming

fastp (Chen 2023) is set to auto-detect Illumina adapters, as well as trim the first 10bp from each read, as past experience shows these first 10bp are more inconsistent than the remainder of the read length.

Note

Appends _L001 to trimmed filenames for downstream QIIME 2 filename parsing requirements.


# Make output directories, if it doesn't exist
mkdir --parents "${output_dir}"

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

# Create arrays of fastq R1 files and sample names
for fastq in ${R1_fastq_pattern}
do
  fastq_array_R1+=("${fastq}")
  R1_names_array+=("$(echo "${fastq}" | awk -F'_' '{print $1"_"$2"_"$3"_"$4"_L001"}')")
done



# Create array of fastq R2 files
for fastq in ${R2_fastq_pattern}
do
  fastq_array_R2+=("${fastq}")
  R2_names_array+=("$(echo "${fastq}" | awk -F"_" '{print $1"_"$2"_"$3"_"$4"_L001"}')")
done

# Create list of fastq files used in analysis
# Create MD5 checksum for reference
if [ ! -f "${output_dir}"/raw-fastq-checksums.md5 ]; then
  for fastq in *.gz
    do
      md5sum "${fastq}" >>"${output_dir}"/raw-fastq-checksums.md5
  done
fi

# Run fastp on files
# Adds JSON report output for downstream usage by MultiQC
for index in "${!fastq_array_R1[@]}"
do
  R1_sample_name=$(echo "${R1_names_array[index]}")
  R2_sample_name=$(echo "${R2_names_array[index]}")
  
  
  ${fastp} \
  --in1 ${fastq_array_R1[index]} \
  --in2 ${fastq_array_R2[index]} \
  --detect_adapter_for_pe \
  --trim_front1 20 \
  --trim_front2 20 \
  --trim_poly_g \
  --thread ${threads} \
  --html "${output_dir}"/"${R1_sample_name}".fastp-trim.report.html \
  --json "${output_dir}"/"${R1_sample_name}".fastp-trim.report.json \
  --out1 "${output_dir}"/"${R1_sample_name}"_R1.fastp-trim.fastq.gz \
  --out2 "${output_dir}"/"${R2_sample_name}"_R2.fastp-trim.fastq.gz \
  2>> "${output_dir}"/"${R1_sample_name}"-fastp.stderr

  # Generate md5 checksums for newly trimmed files
  cd "${output_dir}"
  md5sum "${R1_sample_name}"_R1.fastp-trim.fastq.gz | tee "${R1_sample_name}"_R1.fastp-trim.fastq.gz.md5
  md5sum "${R2_sample_name}"_R2.fastp-trim.fastq.gz | tee "${R2_sample_name}"_R2.fastp-trim.fastq.gz.md5
  
  # Change back to previous directory
  # Directing to /dev/null prevents printing directory each time.
  cd - > /dev/null
done
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4 FastQC/MultiQC on trimmed reads

4.1 Create FastQ list to use across chunks

# Create the fastq list and string
trimmed_fastqs_list <- list.files(path = Sys.getenv("output_dir"), pattern = "\\.fastq\\.gz$", full.names = TRUE)
trimmed_fastqs_string <- paste(trimmed_fastqs_list, collapse = " ")

# Update the environment variable with the actual string
Sys.setenv(trimmed_fastqs_string = trimmed_fastqs_string)

4.2 Run FastQC/MultiQC


# Make output directory if it doesn't exist
mkdir --parents "${output_dir}"

cd "${output_dir}"

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


# Create array of trimmed FastQs
# Access the individual FastQ files from the array
IFS=' ' read -r -a trimmed_fastqs_array <<< "${trimmed_fastqs_string}"

# 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
### NOTE: Do NOT quote trimmed_fastqc_list
${fastqc} \
--threads ${threads} \
--outdir ${output_dir} \
--quiet \
${trimmed_fastqc_list}

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

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

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

${multiqc} --interactive \
${output_dir} \
-o ${output_dir}

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

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

echo "Removing FastQC zip files."
echo ""
rm ${output_dir}/*.zip
echo "FastQC zip files removed."
echo ""
Beginning FastQC on raw reads...

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FastQC on raw reads complete!

Beginning MultiQC on raw FastQC...


/// MultiQC 🔍 v1.28

       file_search | Search path: /home/sam/gitrepos/RobertsLab/project-eDNA-yellow/output/01.00-trimming-fastp-fastqc-multiqc

             fastp | Found 54 reports
            fastqc | Found 108 reports

     write_results | Data        : multiqc_data
     write_results | Report      : multiqc_report.html
           multiqc | MultiQC complete

MultiQC on raw FastQs complete.

Removing FastQC zip files.

FastQC zip files removed.

4.3 List output files

cd "${output_dir}"

ls -lh
total 1.3G
drwxrwxr-x 2 sam sam 4.0K Apr 18 12:48 multiqc_data
-rw-rw-r-- 1 sam sam 8.0M Apr 18 12:48 multiqc_report.html
-rw-rw-r-- 1 sam sam 7.8K Apr 18 12:26 raw-fastq-checksums.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:27 Yell_July18_Site1a_S83_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 489K Apr 18 12:27 Yell_July18_Site1a_S83_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 174K Apr 18 12:27 Yell_July18_Site1a_S83_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 922K Apr 18 12:46 Yell_July18_Site1a_S83_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 9.7M Apr 18 12:27 Yell_July18_Site1a_S83_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:27 Yell_July18_Site1a_S83_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 915K Apr 18 12:46 Yell_July18_Site1a_S83_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  11M Apr 18 12:27 Yell_July18_Site1a_S83_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:27 Yell_July18_Site1a_S83_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:27 Yell_July18_Site1bb_S139_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 488K Apr 18 12:27 Yell_July18_Site1bb_S139_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 174K Apr 18 12:27 Yell_July18_Site1bb_S139_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 922K Apr 18 12:46 Yell_July18_Site1bb_S139_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 7.0M Apr 18 12:27 Yell_July18_Site1bb_S139_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   87 Apr 18 12:27 Yell_July18_Site1bb_S139_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 932K Apr 18 12:46 Yell_July18_Site1bb_S139_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 7.5M Apr 18 12:27 Yell_July18_Site1bb_S139_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   87 Apr 18 12:27 Yell_July18_Site1bb_S139_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:28 Yell_July18_Site1b_S84_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 488K Apr 18 12:28 Yell_July18_Site1b_S84_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 173K Apr 18 12:28 Yell_July18_Site1b_S84_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 919K Apr 18 12:46 Yell_July18_Site1b_S84_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 9.2M Apr 18 12:28 Yell_July18_Site1b_S84_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:28 Yell_July18_Site1b_S84_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 910K Apr 18 12:46 Yell_July18_Site1b_S84_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  10M Apr 18 12:28 Yell_July18_Site1b_S84_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:28 Yell_July18_Site1b_S84_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:28 Yell_July18_Site1cc_S140_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 490K Apr 18 12:28 Yell_July18_Site1cc_S140_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 175K Apr 18 12:28 Yell_July18_Site1cc_S140_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 924K Apr 18 12:46 Yell_July18_Site1cc_S140_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  17M Apr 18 12:28 Yell_July18_Site1cc_S140_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   87 Apr 18 12:28 Yell_July18_Site1cc_S140_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 914K Apr 18 12:46 Yell_July18_Site1cc_S140_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  19M Apr 18 12:28 Yell_July18_Site1cc_S140_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   87 Apr 18 12:28 Yell_July18_Site1cc_S140_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:29 Yell_July18_Site1c_S85_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 487K Apr 18 12:29 Yell_July18_Site1c_S85_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 173K Apr 18 12:29 Yell_July18_Site1c_S85_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 932K Apr 18 12:46 Yell_July18_Site1c_S85_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 6.8M Apr 18 12:29 Yell_July18_Site1c_S85_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:29 Yell_July18_Site1c_S85_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 937K Apr 18 12:46 Yell_July18_Site1c_S85_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 7.4M Apr 18 12:29 Yell_July18_Site1c_S85_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:29 Yell_July18_Site1c_S85_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:29 Yell_July18_Site1dd_S141_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 489K Apr 18 12:29 Yell_July18_Site1dd_S141_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 175K Apr 18 12:29 Yell_July18_Site1dd_S141_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 931K Apr 18 12:46 Yell_July18_Site1dd_S141_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  19M Apr 18 12:29 Yell_July18_Site1dd_S141_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   87 Apr 18 12:29 Yell_July18_Site1dd_S141_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 936K Apr 18 12:46 Yell_July18_Site1dd_S141_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  19M Apr 18 12:29 Yell_July18_Site1dd_S141_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   87 Apr 18 12:29 Yell_July18_Site1dd_S141_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:29 Yell_July18_Site1d_S86_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 488K Apr 18 12:29 Yell_July18_Site1d_S86_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 174K Apr 18 12:29 Yell_July18_Site1d_S86_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 922K Apr 18 12:46 Yell_July18_Site1d_S86_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 9.5M Apr 18 12:29 Yell_July18_Site1d_S86_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:29 Yell_July18_Site1d_S86_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 935K Apr 18 12:46 Yell_July18_Site1d_S86_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  11M Apr 18 12:29 Yell_July18_Site1d_S86_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:29 Yell_July18_Site1d_S86_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:30 Yell_July18_Site1ee_S142_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 489K Apr 18 12:30 Yell_July18_Site1ee_S142_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 174K Apr 18 12:30 Yell_July18_Site1ee_S142_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 944K Apr 18 12:46 Yell_July18_Site1ee_S142_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  12M Apr 18 12:30 Yell_July18_Site1ee_S142_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   87 Apr 18 12:30 Yell_July18_Site1ee_S142_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 952K Apr 18 12:46 Yell_July18_Site1ee_S142_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  11M Apr 18 12:30 Yell_July18_Site1ee_S142_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   87 Apr 18 12:30 Yell_July18_Site1ee_S142_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:30 Yell_July18_Site1e_S91_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 484K Apr 18 12:30 Yell_July18_Site1e_S91_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 170K Apr 18 12:30 Yell_July18_Site1e_S91_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 921K Apr 18 12:46 Yell_July18_Site1e_S91_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 1.6M Apr 18 12:30 Yell_July18_Site1e_S91_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:30 Yell_July18_Site1e_S91_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 930K Apr 18 12:46 Yell_July18_Site1e_S91_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 1.7M Apr 18 12:30 Yell_July18_Site1e_S91_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:30 Yell_July18_Site1e_S91_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:30 Yell_July18_Site1f_S92_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 489K Apr 18 12:30 Yell_July18_Site1f_S92_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 175K Apr 18 12:30 Yell_July18_Site1f_S92_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 904K Apr 18 12:46 Yell_July18_Site1f_S92_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  11M Apr 18 12:30 Yell_July18_Site1f_S92_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:30 Yell_July18_Site1f_S92_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 918K Apr 18 12:46 Yell_July18_Site1f_S92_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  12M Apr 18 12:30 Yell_July18_Site1f_S92_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:30 Yell_July18_Site1f_S92_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:31 Yell_July18_Site1g_S93_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 488K Apr 18 12:31 Yell_July18_Site1g_S93_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 174K Apr 18 12:31 Yell_July18_Site1g_S93_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 914K Apr 18 12:46 Yell_July18_Site1g_S93_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 9.7M Apr 18 12:31 Yell_July18_Site1g_S93_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:31 Yell_July18_Site1g_S93_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 921K Apr 18 12:46 Yell_July18_Site1g_S93_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  11M Apr 18 12:31 Yell_July18_Site1g_S93_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:31 Yell_July18_Site1g_S93_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:31 Yell_July18_Site1h_S94_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 486K Apr 18 12:31 Yell_July18_Site1h_S94_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 172K Apr 18 12:31 Yell_July18_Site1h_S94_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 916K Apr 18 12:46 Yell_July18_Site1h_S94_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 3.5M Apr 18 12:31 Yell_July18_Site1h_S94_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:31 Yell_July18_Site1h_S94_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 932K Apr 18 12:46 Yell_July18_Site1h_S94_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 3.8M Apr 18 12:31 Yell_July18_Site1h_S94_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:31 Yell_July18_Site1h_S94_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:31 Yell_July18_Site1J_S99_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 482K Apr 18 12:31 Yell_July18_Site1J_S99_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 169K Apr 18 12:31 Yell_July18_Site1J_S99_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 909K Apr 18 12:46 Yell_July18_Site1J_S99_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 1.1M Apr 18 12:31 Yell_July18_Site1J_S99_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:31 Yell_July18_Site1J_S99_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 930K Apr 18 12:46 Yell_July18_Site1J_S99_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 851K Apr 18 12:31 Yell_July18_Site1J_S99_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   85 Apr 18 12:31 Yell_July18_Site1J_S99_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:31 Yell_July18_Site1k_S100_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 487K Apr 18 12:31 Yell_July18_Site1k_S100_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 173K Apr 18 12:31 Yell_July18_Site1k_S100_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 919K Apr 18 12:46 Yell_July18_Site1k_S100_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 7.1M Apr 18 12:31 Yell_July18_Site1k_S100_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   86 Apr 18 12:31 Yell_July18_Site1k_S100_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 929K Apr 18 12:46 Yell_July18_Site1k_S100_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 7.0M Apr 18 12:31 Yell_July18_Site1k_S100_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   86 Apr 18 12:31 Yell_July18_Site1k_S100_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:32 Yell_July18_Site1L_S101_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 486K Apr 18 12:32 Yell_July18_Site1L_S101_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 171K Apr 18 12:32 Yell_July18_Site1L_S101_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 921K Apr 18 12:46 Yell_July18_Site1L_S101_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 2.6M Apr 18 12:32 Yell_July18_Site1L_S101_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   86 Apr 18 12:32 Yell_July18_Site1L_S101_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 931K Apr 18 12:46 Yell_July18_Site1L_S101_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 3.0M Apr 18 12:32 Yell_July18_Site1L_S101_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   86 Apr 18 12:32 Yell_July18_Site1L_S101_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:32 Yell_July18_Site1m_S102_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 488K Apr 18 12:32 Yell_July18_Site1m_S102_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 174K Apr 18 12:32 Yell_July18_Site1m_S102_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 916K Apr 18 12:46 Yell_July18_Site1m_S102_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam 9.9M Apr 18 12:32 Yell_July18_Site1m_S102_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   86 Apr 18 12:32 Yell_July18_Site1m_S102_L001_R1.fastp-trim.fastq.gz.md5
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-rw-rw-r-- 1 sam sam   86 Apr 18 12:32 Yell_July18_Site1m_S102_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:32 Yell_July18_Site1n_S103_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 490K Apr 18 12:32 Yell_July18_Site1n_S103_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 175K Apr 18 12:32 Yell_July18_Site1n_S103_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 919K Apr 18 12:46 Yell_July18_Site1n_S103_L001_R1.fastp-trim_fastqc.html
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-rw-rw-r-- 1 sam sam   86 Apr 18 12:32 Yell_July18_Site1n_S103_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 906K Apr 18 12:46 Yell_July18_Site1n_S103_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  15M Apr 18 12:32 Yell_July18_Site1n_S103_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   86 Apr 18 12:32 Yell_July18_Site1n_S103_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:33 Yell_July18_Site1o_S104_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 489K Apr 18 12:33 Yell_July18_Site1o_S104_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 174K Apr 18 12:33 Yell_July18_Site1o_S104_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 917K Apr 18 12:46 Yell_July18_Site1o_S104_L001_R1.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  12M Apr 18 12:33 Yell_July18_Site1o_S104_L001_R1.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   86 Apr 18 12:33 Yell_July18_Site1o_S104_L001_R1.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 924K Apr 18 12:46 Yell_July18_Site1o_S104_L001_R2.fastp-trim_fastqc.html
-rw-rw-r-- 1 sam sam  13M Apr 18 12:33 Yell_July18_Site1o_S104_L001_R2.fastp-trim.fastq.gz
-rw-rw-r-- 1 sam sam   86 Apr 18 12:33 Yell_July18_Site1o_S104_L001_R2.fastp-trim.fastq.gz.md5
-rw-rw-r-- 1 sam sam 2.1K Apr 18 12:33 Yell_July18_Site1p_S105_L001-fastp.stderr
-rw-rw-r-- 1 sam sam 491K Apr 18 12:33 Yell_July18_Site1p_S105_L001.fastp-trim.report.html
-rw-rw-r-- 1 sam sam 177K Apr 18 12:33 Yell_July18_Site1p_S105_L001.fastp-trim.report.json
-rw-rw-r-- 1 sam sam 916K Apr 18 12:47 Yell_July18_Site1p_S105_L001_R1.fastp-trim_fastqc.html
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RESULTS

Trimming looks as expected. Due to the heterogeneous nature of eDNA, we don’t see the usual “smooth” lines across the length of the reads when viewing the “Per Base Sequence Content” plots from FastQC/MultiQC. As such, we’ll proceed with QIIME 2 18s analyses of these samples.

Screenshot of example MultiQC Per Base Sequence Content showing expected uneven sequences, due to heterogenous content of eDNA.

Screenshot of example MultiQC Per Base Sequence Content showing expected uneven sequences, due to heterogenous content of eDNA.

REFERENCES

Chen, Shifu. 2023. “Ultrafast One-Pass FASTQ Data Preprocessing, Quality Control, and Deduplication Using Fastp.” iMeta 2 (2). https://doi.org/10.1002/imt2.107.
Ewels, Philip, Måns Magnusson, Sverker Lundin, and Max Käller. 2016. “MultiQC: Summarize Analysis Results for Multiple Tools and Samples in a Single Report.” Bioinformatics 32 (19): 3047–48. https://doi.org/10.1093/bioinformatics/btw354.