Transcriptome Assembly - C.bairdi All RNAseq Data Without Taxonomic Filters with Trinity on Mox

2020
Miscellaneous
Author

Sam White

Published

May 2, 2020

Steven asked that I assemble an unfiltered (i.e. no taxonomic selection) transcriptome with all of our C.bairdi RNAseq data (see the FastQ list file linked in the Results section below). A de novo assembly was run using Trinity on Mox. It should be noted that this assembly is a mixture of stranded/non-stranded library preps.

SBATCH Script (GitHub):

#!/bin/bash
## Job Name
#SBATCH --job-name=trinity_cbai
## Allocation Definition
#SBATCH --account=srlab
#SBATCH --partition=srlab
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=9-00:00:00
## 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/20200502_cbai_trinity_all_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/srlab/sam/data/C_bairdi/RNAseq
transcriptome_dir=/gscratch/srlab/sam/data/C_bairdi/transcriptomes
threads=28
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*fastp-trim*.fq.gz)

# Create array of fastq R2 files
R2_array=("${reads_dir}"/*_R2*fastp-trim*.fq.gz)

# Create list of fastq files used in analysis
## Uses parameter substitution to strip leading path from filename
for fastq in "${reads_dir}"/*fastp-trim*.fq.gz
do
  echo "${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
## Not running as "stranded", due to mix of library types
${trinity_dir}/Trinity \
--seqType fq \
--max_memory 500G \
--CPU ${threads} \
--left "${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 sequence lengths file (used for differential gene expression)
${trinity_dir}/util/misc/fasta_seq_length.pl \
trinity_out_dir/"${fasta_name}" \
> trinity_out_dir/"${fasta_name}".seq_lens

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

# Copy files to transcriptome directory
rsync -av \
trinity_out_dir/"${fasta_name}"* \
${transcriptome_dir}

RESULTS

There were some hiccups (Mox crashes, weird Trinity error that interrupted job), but overall, it took ~4 days of actual run time.

cbai Trinity all RNAseq runtime

Output folder:

FastQ list (text):

FastA (904MB):

NOTE: The transcriptome will be referred to as cbai_transcriptome_v2.0.fasta and has been added to our Genomic Resources wiki.

Trinity gene trans map (text; useful for downstream gene expression/annotation with Trinity/Trinotate):

Trinity FastA sequence lengths file (text; useful for downstream gene expression/annotation with Trinity/Trinotate):

Assemby stats (text):

################################
## Counts of transcripts, etc.
################################
Total trinity 'genes':  783006
Total trinity transcripts:  1412254
Percent GC: 45.41

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

    Contig N10: 3733
    Contig N20: 2571
    Contig N30: 1863
    Contig N40: 1285
    Contig N50: 811

    Median contig length: 325
    Average contig: 579.92
    Total assembled bases: 819000346


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

    Contig N10: 3093
    Contig N20: 1768
    Contig N30: 933
    Contig N40: 576
    Contig N50: 431

    Median contig length: 285
    Average contig: 434.16
    Total assembled bases: 339947966