I had previously attempted to compare all of our C.bairdi transcriptome assemblies using DETONATE on 20200601, but, due to hitting time limits on Mox, failed to successfully get the analysis to complete. I realized that the limiting factor was performing FastQ alignments, so I decided to run this step independently to see if I could at least get that step resolved. DETONATE (rsem-eval) will accept BAM files as input, so I’m hoping I can power through this alignment step and then provided DETONATE (rsem-eval) with the BAM files.

I ran bowtie2 on Mox using the alignment settings described in the DETONATE (rsem-eval) documentation (see SBATCH script below for actual bowtie2 parameters).

SBATCH script (GitHub):

20201224_cbai_bowtie2_transcriptomes_alignments.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=20201224_cbai_bowtie2_transcriptomes_alignments
## Allocation Definition
#SBATCH --account=srlab
#SBATCH --partition=srlab
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=15-00:00:00
## Memory per node
#SBATCH --mem=500G
##turn on e-mail notification
#SBATCH --mail-type=ALL
#SBATCH --mail-user=samwhite@uw.edu
## Specify the working directory for this job
#SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20201224_cbai_bowtie2_transcriptomes_alignments
# This is a script to generate BAM files for use in DETONATE's
# rsem-eval program to compare C.bairdi transcriptome assembly "qualities".

###################################################################################
# These variables need to be set by user

# Assign Variables
reads_dir=/gscratch/srlab/sam/data/C_bairdi/RNAseq
transcriptomes_dir=/gscratch/srlab/sam/data/C_bairdi/transcriptomes
threads=28
mem_per_thread=10G

# Program paths
bowtie2_dir="/gscratch/srlab/programs/bowtie2-2.4.2-linux-x86_64"
samtools="/gscratch/srlab/programs/samtools-1.10/samtools"

# Array of the various comparisons to evaluate
# Each condition in each comparison should be separated by a "-"
transcriptomes_array=(
"${transcriptomes_dir}"/cbai_transcriptome_v1.0.fasta \
"${transcriptomes_dir}"/cbai_transcriptome_v1.5.fasta \
"${transcriptomes_dir}"/cbai_transcriptome_v1.6.fasta \
"${transcriptomes_dir}"/cbai_transcriptome_v1.7.fasta \
"${transcriptomes_dir}"/cbai_transcriptome_v2.0.fasta \
"${transcriptomes_dir}"/cbai_transcriptome_v2.1.fasta \
"${transcriptomes_dir}"/cbai_transcriptome_v3.0.fasta \
"${transcriptomes_dir}"/cbai_transcriptome_v3.1.fasta
)


###################################################################################

# Exit script if any command fails
set -e

# Load Python Mox module for Python module availability
module load intel-python3_2017


# Programs array
declare -A programs_array
programs_array=(
[bowtie2]="${bowtie2_dir}/bowtie2" \
[bowtie2_build]="${bowtie2_dir}/bowtie2-build" \
[samtools_index]="${samtools} index" \
[samtools_sort]="${samtools} sort" \
[samtools_view]="${samtools} view"
)




# Loop through each comparison
for transcriptome in "${!transcriptomes_array[@]}"
do

  ## Inititalize arrays
  R1_array=()
  R2_array=()
  reads_array=()

  # Variables
  R1_list=""
  R2_list=""

  transcriptome_name="${transcriptomes_array[$transcriptome]##*/}"

  # Capture FastA checksums for verification
  echo "Generating checksum for ${transcriptome_name}"
  md5sum "${transcriptomes_array[transcriptome]}" >> fasta.checksums.md5
  echo "Finished generating checksum for ${transcriptome_name}"
  echo ""

    if [[ "${transcriptome_name}" == "cbai_transcriptome_v1.0.fasta" ]]; then

    reads_array=("${reads_dir}"/20200[15][13][138]*megan*.fq)

    # Create array of fastq R1 files
    R1_array=("${reads_dir}"/20200[15][13][138]*megan*R1.fq)

    # Create array of fastq R2 files
    R2_array=("${reads_dir}"/20200[15][13][138]*megan*R2.fq)



  elif [[ "${transcriptome_name}" == "cbai_transcriptome_v1.5.fasta" ]]; then

    reads_array=("${reads_dir}"/20200[145][13][138]*megan*.fq)

    # Create array of fastq R1 files
    R1_array=("${reads_dir}"/20200[145][13][138]*megan*R1.fq)

    # Create array of fastq R2 files
    R2_array=("${reads_dir}"/20200[145][13][138]*megan*R2.fq)

  elif [[ "${transcriptome_name}" == "cbai_transcriptome_v1.6.fasta" ]]; then

    reads_array=("${reads_dir}"/*megan*.fq)

    # Create array of fastq R1 files
    R1_array=("${reads_dir}"/*megan*R1.fq)

    # Create array of fastq R2 files
    R2_array=("${reads_dir}"/*megan*R2.fq)

  elif [[ "${transcriptome_name}" == "cbai_transcriptome_v1.7.fasta" ]]; then

    reads_array=("${reads_dir}"/20200[145][13][189]*megan*.fq)

    # Create array of fastq R1 files
    R1_array=("${reads_dir}"/20200[145][13][189]*megan*R1.fq)

    # Create array of fastq R2 files
    R2_array=("${reads_dir}"/20200[145][13][189]*megan*R2.fq)

  elif [[ "${transcriptome_name}" == "cbai_transcriptome_v2.0.fasta" ]] \
  || [[ "${transcriptome_name}" == "cbai_transcriptome_v2.1.fasta" ]]; then

    reads_array=("${reads_dir}"/*fastp-trim*.fq)

    # Create array of fastq R1 files
    R1_array=("${reads_dir}"/*R1*fastp-trim*.fq)

    # Create array of fastq R2 files
    R2_array=("${reads_dir}"/*R2*fastp-trim*.fq)

  elif [[ "${transcriptome_name}" == "cbai_transcriptome_v3.0.fasta" ]] \
  || [[ "${transcriptome_name}" == "cbai_transcriptome_v3.1.fasta" ]]; then

    reads_array=("${reads_dir}"/*fastp-trim*20[12][09][01][24]1[48]*.fq)

    # Create array of fastq R1 files
    R1_array=("${reads_dir}"/*R1*fastp-trim*20[12][09][01][24]1[48]*.fq)

    # Create array of fastq R2 files
    R2_array=("${reads_dir}"/*R2*fastp-trim*20[12][09][01][24]1[48]*.fq)


  fi

  # Create list of fastq files used in analysis
  ## Uses parameter substitution to strip leading path from filename
  printf "%s\n" "${reads_array[@]##*/}" >> "${transcriptome_name}".fastq.list.txt

  # Create comma-separated lists of FastQ reads
  R1_list=$(echo "${R1_array[@]}" | tr " " ",")
  R2_list=$(echo "${R2_array[@]}" | tr " " ",")

  # Build Bowtie2 index
  # Transcriptome name is used as index basename
  ${programs_array[bowtie2_build]} \
  --threads ${threads} \
  ${transcriptomes_array[$transcriptome]} \
  ${transcriptome_name}

  # Run rsem-eval
  # Use bowtie2 and paired-end options
  # Uses settings specified for use with DETONATE
  # and for paired end reads when using DETONATE.
  ${programs_array[bowtie2]} \
  -x ${transcriptome_name} \
  -S ${transcriptome_name}.sam \
  --threads ${threads} \
  -1 ${R1_list} \
  -2 ${R2_list} \
  --sensitive \
  --dpad 0 \
  --gbar 99999999 \
  --mp 1,1 \
  --np 1 \
  --score-min L,0,-0.1 \
  --no-mixed \
  --no-discordant

  # Convert SAM to sorted BAM
  #
  ${programs_array[samtools_view]} \
  -b \
  ${transcriptome_name}.sam \
  | ${programs_array[samtools_sort]} \
  -m ${mem_per_thread} \
  --threads ${threads} \
  -o ${transcriptome_name}.sorted.bam \
  -

  # Capture BAM checksums for verification
  echo "Generating checksum for ${transcriptome_name}.sorted.bam"
  md5sum ${transcriptome_name}.sorted.bam >> bam.checksums.md5
  echo "Finished generating checksum for ${transcriptome_name}.sorted.bam"
  echo ""

done

# Remove leftover SAM files
rm *.sam

# Capture program options
echo "Logging program options..."
for program in "${!programs_array[@]}"
do
    {
  echo "Program options for ${program}: "
    echo ""
  # Handle samtools help menus
  if [[ "${program}" == "samtools_index" ]] \
  || [[ "${program}" == "samtools_sort" ]] \
  || [[ "${program}" == "samtools_view" ]]
  then
    ${programs_array[$program]}
  fi
    ${programs_array[$program]} -h
    echo ""
    echo ""
    echo "----------------------------------------------"
    echo ""
    echo ""
} &>> program_options.log || true

  # If MultiQC is in programs_array, copy the config file to this directory.
  if [[ "${program}" == "multiqc" ]]; then
    cp --preserve ~/.multiqc_config.yaml multiqc_config.yaml
  fi
done

echo ""
echo "Finished logging program options."
echo ""

echo ""
echo "Logging system PATH."
# Document programs in PATH (primarily for program version ID)
{
date
echo ""
echo "System PATH for $SLURM_JOB_ID"
echo ""
printf "%0.s-" {1..10}
echo "${PATH}" | tr : \\n
} >> system_path.log

echo "Finished logging system PATH"

RESULTS

Run time was ~2.5 days (not counting some weird issues with Mox):

~Runtime for bowtie2 alignments on Mox

These will be used in a subsequent analysis using DETONATE (rsem-eval) to generate a score for each transcriptome. This score can be utilized to help compare the transcriptomes to identify which is the best assembly.

Important output files are linked below (BAMs, checksums, list of FastQs for each alignment).

Output folder:

20201224_cbai_bowtie2_transcriptomes_alignments/
- MD5 checksum files (text) for input FastAs and resulting BAMs:
  - bam.checksums.md5
  - fasta.checksums.md5
- cbai_transcriptome_v1.0 files:
  - bowtie2 BAM file:
    - cbai_transcriptome_v1.0.fasta.sorted.bam
  - List of FastQs used for alignment (text):
    - cbai_transcriptome_v1.0.fasta.fastq.list.txt
- cbai_transcriptome_v1.5 files:
  - [bowtie2](https://gith - List of FastQs used for alignment (text):
    - cbai_transcriptome_v1.7.fasta.fastq.list.txtub.com/BenLangmead/bowtie2) BAM file:
    - cbai_transcriptome_v1.5.fasta.sorted.bam
  - List of FastQs used for alignment (text):
    - cbai_transcriptome_v1.5.fasta.fastq.list.txt
- cbai_transcriptome_v1.6 files:
  - bowtie2 BAM file:
    - cbai_transcriptome_v1.6.fasta.sorted.bam
  - List of FastQs used for alignment (text):
    - cbai_transcriptome_v1.6.fasta.fastq.list.txt
- cbai_transcriptome_v1.7 files:
  - bowtie2 BAM file:
    - cbai_transcriptome_v1.7.fasta.sorted.bam
  - List of FastQs used for alignment (text):
    - cbai_transcriptome_v1.7.fasta.fastq.list.txt
- cbai_transcriptome_v2.0 files:
  - bowtie2 BAM file:
    - cbai_transcriptome_v2.0.fasta.sorted.bam
  - List of FastQs used for alignment (text):
    - cbai_transcriptome_v1.7.fasta.fastq.list.txt
- cbai_transcriptome_v2.1 files:
  - bowtie2 BAM file:
    - cbai_transcriptome_v2.1.fasta.sorted.bam
  - List of FastQs used for alignment (text):
    - cbai_transcriptome_v2.1.fasta.fastq.list.txt
- cbai_transcriptome_v3.0 files:
  - bowtie2 BAM file:
    - cbai_transcriptome_v3.0.fasta.sorted.bam
  - List of FastQs used for alignment (text):
    - cbai_transcriptome_v3.0.fasta.fastq.list.txt
- cbai_transcriptome_v3.1 files:
  - bowtie2 BAM file:
    - cbai_transcriptome_v3.1.fasta.sorted.bam
  - List of FastQs used for alignment (text):
    - cbai_transcriptome_v3.1.fasta.fastq.list.txt