Clarified with Steven an approach for tackling multi-condition comparisons (see this GitHub Issue). As such, I need to have individual transcript abundances for each sample from the 2020 Genewiz RNAseq data before I can proceed. So, I ran salmon (v1.2.1) to perform an alignment-free set of transcript abundances. It’s ridiculously fast, btw…

This was run on Mox and used the C.bairdi-specific reads that were extracted using MEGAN6 on 202020330.

SBATCH script (GitHub):

20200429_cbai_salmon_2020GW_transcript_abundances.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=cbai_DEG_basic
## Allocation Definition
#SBATCH --account=coenv
#SBATCH --partition=coenv
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=10-00:00:00
## Memory per node
#SBATCH --mem=120G
##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/20200429_cbai_salmon_2020GW_transcript_abundances

# Script to generate set of transcript abundances for all C.bairdi Genewiz 2020 data.
#
# C.bairdi-specific reads were extracted with MEGAN6:
# https://robertslab.github.io/sams-notebook/posts/2020/2020-03-30-RNAseq-Reads-Extractions---C.bairdi-Taxonomic-Reads-Extractions-with-MEGAN6-on-swoose/
#
# Transcriptome was produced here: https://robertslab.github.io/sams-notebook/posts/2020/2020-03-30-Transcriptome-Assembly---C.bairdi-with-MEGAN6-Taxonomy-specific-Reads-with-Trinity-on-Mox/
# Transcriptome is the same as: cbai_transcriptome_v1.5.fasta
#
# Salmon index generated during a previous gene expression analysis:
# https://robertslab.github.io/sams-notebook/posts/2020/2020-04-22-Gene-Expression---C.bairdi-Pairwise-DEG-Comparisons-with-2019-RNAseq-using-Trinity-Salmon-EdgeR-on-Mox/



###################################################################################################################
# BEGIN USER SETTINGS


# Programs array
declare -A programs_array
programs_array=([salmon]="/gscratch/srlab/programs/salmon-1.2.1_linux_x86_64/bin/salmon")

## Designate input files and locations
fastq_dir="/gscratch/srlab/sam/data/C_bairdi/RNAseq/"
salmon_index="/gscratch/srlab/sam/data/C_bairdi/transcriptomes/20200408.C_bairdi.megan.Trinity.fasta.salmon.idx"

# Set number of CPU threads
# Salmon default is 56 threads - so not needed
# threads=28

# END USER SETTINGS
####################################################################################################################

# Exit script if any command fails
set -e

# Load Python Mox module for Python module availability

module load intel-python3_2017

# 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

# Caputure working directory
#wd="$(pwd)"


# Capture program options
## NOTE: This particular instance is specific to salmon!
for program in "${!programs_array[@]}"
do
    {
  echo "Program options for ${programs_array[$program]}: "
    echo ""
    ${programs_array[$program]} quant --help
    echo ""
    ${programs_array[$program]} quant --help-reads
    echo ""
    echo "----------------------------------------------"
    echo ""
    echo ""
  } &>> program_options.log || true
done

# Populate array with FastQ files
reads_array=("${fastq_dir}"*.fq)

# Loop through read pairs
# Increment by 2 to process next pair of FastQ files
for (( i=0; i<${#reads_array[@]} ; i+=2 ))
do

    # Create list of FastQ files used
    {
    echo "${reads_array[i]}"
    echo "${reads_array[i+1]}"
} >> fastq-list.txt

  # Strip path and save just sample number
    # Expects sample name to be like:
    # 20200413.C_bairdi.359.D12.infected.ambient.megan_R2.fq
    # Will pull out '359'
  sample=$(echo ${reads_array[i]##*/} | awk -F"." '{print $3}')

    # Run salmon
    # Library type (stranded or not) is set to auto (A)
    ${programs_array[salmon]} quant \
    --index ${salmon_index} \
    --libType A \
    --validateMappings \
    --output "${sample}"_quant \
    -1 ${reads_array[i]} \
    -2 ${reads_array[i+1]}
done

RESULTS

Extremely fast, ~3mins:

Output folder:

20200429_cbai_salmon_2020GW_transcript_abundances

Here are links to the individual quants files:

RESULTS

Sample 72

Sample 73

Sample 113

Sample 118

Sample 127

Sample 132

Sample 151

Sample 173

Sample 178

Sample 221

Sample 222

Sample 254

Sample 272

Sample 280

Sample 294

Sample 334

Sample 349

Sample 359

Sample 425

Sample 427

Sample 445

Sample 463

Sample 481

Sample 485