Earlier today, I trimmed our existing C.bairdi RNAseq data, as part of producing generating a transcriptome (per this GitHub issue). After trimming, I performed a de novo assembly using Trinity (v2.9.0) with the stranded library option (--SS_lib_type RF) on Mox.

List of input files used (text):

20191218_cbai_trinity_RNAseq/fastq.list.tx

SBATCH script (GitHub):

20191218_cbai_trinity_RNAseq.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=trin_cbai
## Allocation Definition
#SBATCH --account=srlab
#SBATCH --partition=srlab
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=30-00:00:00
## 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/20191218_cbai_trinity_RNAseq

# Exit script if a 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

# User-defined variables
reads_dir=/gscratch/scrubbed/samwhite/outputs/20191218_cbai_fastp_RNAseq_trimming
threads=27
assembly_stats=assembly_stats.txt
timestamp=$(date +%Y%m%d)
fasta_name="${timestamp}.C_bairdi.Trinity.fasta"

# Paths to programs
trinity_dir="/gscratch/srlab/programs/trinityrnaseq-v2.9.0"
samtools="/gscratch/srlab/programs/samtools-1.10/samtools"


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

# Variables for R1/R2 lists
R1_list=""
R2_list=""

# Create array of fastq R1 files
R1_array=(${reads_dir}/*_R1_*.gz)

# Create array of fastq R2 files
R2_array=(${reads_dir}/*_R2_*.gz)

# Create list of fastq files used in analysis
## Uses parameter substitution to strip leading path from filename
for fastq in ${reads_dir}/*.gz
do
  echo "${fastq##*/}" >> fastq.list.txt
done

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


# Run Trinity using "stranded" setting (--SS_lib_type)
${trinity_dir}/Trinity \
--seqType fq \
--max_memory 500G \
--CPU ${threads} \
--SS_lib_type RF \
--left "${R1_list}" \
--right "${R2_list}"

# Rename generic assembly FastA
mv trinity_out_dir/Trinity.fasta trinity_out_dir/${fasta_name}

# Assembly stats
${trinity_dir}/util/TrinityStats.pl trinity_out_dir/${fasta_name} \
> ${assembly_stats}

# Create gene map files
${trinity_dir}/util/support_scripts/get_Trinity_gene_to_trans_map.pl \
trinity_out_dir/${fasta_name} \
> trinity_out_dir/${fasta_name}.gene_trans_map

# Create FastA index
${samtools} faidx \
trinity_out_dir/${fasta_name}

RESULTS

This ran relatively quickly (~14hrs), but the Mox email system appeared to be significantly delayed (~8rs difference between email notifications and actual start/stop times of the job):

Output folder:

20191218_cbai_trinity_RNAseq/

Trinity FastA:

20191218_cbai_trinity_RNAseq/trinity_out_dir/20191218.C_bairdi.Trinity.fasta

Trinity FastA index (via samtools):

20191218_cbai_trinity_RNAseq/trinity_out_dir/20191218.C_bairdi.Trinity.fasta.fai

Trinity gene trans map:

20191218_cbai_trinity_RNAseq/trinity_out_dir/20191218.C_bairdi.Trinity.fasta.gene_trans_map

Trinity assembly stats (txt):

20191218_cbai_trinity_RNAseq/assembly_stats.txt



################################
## Counts of transcripts, etc.
################################
Total trinity 'genes':  110785
Total trinity transcripts:  313589
Percent GC: 46.11

########################################
Stats based on ALL transcript contigs:
########################################

    Contig N10: 4395
    Contig N20: 3382
    Contig N30: 2773
    Contig N40: 2337
    Contig N50: 1961

    Median contig length: 689
    Average contig: 1146.98
    Total assembled bases: 359680329


#####################################################
## Stats based on ONLY LONGEST ISOFORM per 'GENE':
#####################################################

    Contig N10: 4529
    Contig N20: 3430
    Contig N30: 2780
    Contig N40: 2276
    Contig N50: 1821

    Median contig length: 405
    Average contig: 882.36
    Total assembled bases: 97752083