Ran a de novo assembly on our HiSeq and NovaSeq data from Hollie’s juvenile EPI 124 sample “ambient OA”. This was done for Christian to use in some long, non-coding RNA (lncRNA) analysis.

NovaSeq data had been previously trimmed.

Trimming of the HiSeq data was performed via Trinity, using the --trimmomatic option.

SBATCH script (GitHub):

20190409_trinity_pgen_EPI124_RNAseq.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=trin_epi124
## Allocation Definition
#SBATCH --account=coenv
#SBATCH --partition=coenv
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=30-00:00:00
## 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 --workdir=/gscratch/scrubbed/samwhite/outputs/20190409_trinity_pgen_EPI124_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 >> system_path.log
echo "" >> system_path.log
echo "System PATH for $SLURM_JOB_ID" >> system_path.log
echo "" >> system_path.log
printf "%0.s-" {1..10} >> system_path.log
echo ${PATH} | tr : \\n >> system_path.log

# User-defined variables
reads_dir=/gscratch/scrubbed/samwhite/data/P_generosa/RNAseq/epi_124
threads=28
assembly_stats=assembly_stats.txt

# Paths to programs
trinity_dir="/gscratch/srlab/programs/Trinity-v2.8.3"
samtools="/gscratch/srlab/programs/samtools-1.9/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
${trinity_dir}/Trinity \
--trimmomatic \
--seqType fq \
--max_memory 120G \
--CPU ${threads} \
--left \
${R1_list} \
--right \
${R2_list}

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

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

# Create FastA index
${samtools} faidx \
trinity_out_dir/Trinity.fasta

RESULTS

Output folder:

Trinity FastA:

20190409_trinity_pgen_EPI124_RNAseq/trinity_out_dir/Trinity.fasta

Trinity FastA index file:

20190409_trinity_pgen_EPI124_RNAseq/trinity_out_dir/Trinity.fasta.fai

Trinity Gene Trans Map file:

20190409_trinity_pgen_EPI124_RNAseq/trinity_out_dir/Trinity.fasta.gene_trans_map

Assembly stats (text):

20190409_trinity_pgen_EPI124_RNAseq/assembly_stats.txt

################################
## Counts of transcripts, etc.
################################
Total trinity 'genes':  255277
Total trinity transcripts:  421670
Percent GC: 35.84

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

    Contig N10: 5723
    Contig N20: 3912
    Contig N30: 2887
    Contig N40: 2172
    Contig N50: 1596

    Median contig length: 444
    Average contig: 877.70
    Total assembled bases: 370100426


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

    Contig N10: 4624
    Contig N20: 3006
    Contig N30: 2144
    Contig N40: 1521
    Contig N50: 1060

    Median contig length: 378
    Average contig: 691.20

List of input FastQs (text):

20190409_trinity_pgen_EPI124_RNAseq/fastq.list.txt

Geoduck-juvenile-ambient-exposure-RNA-EPI-124-1_S7_L001_R1_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-1_S7_L001_R2_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-2_S15_L002_R1_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-2_S15_L002_R2_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-3_S23_L003_R1_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-3_S23_L003_R2_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-4_S31_L004_R1_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-4_S31_L004_R2_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-5_S39_L005_R1_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-5_S39_L005_R2_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-6_S47_L006_R1_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-6_S47_L006_R2_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-7_S55_L007_R1_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-7_S55_L007_R2_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-8_S63_L008_R1_001.fastq.gz
Geoduck-juvenile-ambient-exposure-RNA-EPI-124-8_S63_L008_R2_001.fastq.gz
NR019_S7_L001_R1_001_val_1_val_1.fq.gz
NR019_S7_L001_R2_001_val_2_val_2.fq.gz
NR019_S7_L002_R1_001_val_1_val_1.fq.gz
NR019_S7_L002_R2_001_val_2_val_2.fq.gz