For completeness sake, I wanted to create an additional C.bairdi transcriptome assembly that consisted of Arthropoda only sequences from just pooled RNAseq data (since I recently generated a similar assembly without taxonomically filtered reads on 20200518). This constitutes samples we have designated: 2018, 2019, 2020-UW. A de novo assembly was run using Trinity on Mox. Since all pooled RNAseq libraries were stranded, I added this option to Trinity command.

The resulting assembly will be referred to as:

• cbai_transcriptome_v1.7.fasta

SBATCH script (GitHub):

• 20200527_cbai_trinity_arthropoda_pooled_RNAseq.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=trinity_cbai_v1.7
## 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/20200527_cbai_trinity_arthropoda_pooled_RNAseq


### Trinity de novo assembly of all pooled C.bairdi Arthropoda-only RNAseq data.
### Includes "descriptor_1" short-hand of: 2020-UW, 2019, 2018.
### See fastq.list.txt file for list of input files used for assembly.

# 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
fasta_name="cbai_transcriptome_v1.7.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}"/*3[80][804]*R1.fq)

# Create array of fastq R2 files
R2_array=("${reads_dir}"/*3[80][804]*R2.fq)

# Create list of fastq files used in analysis
## Uses parameter substitution to strip leading path from filename
for fastq in "${reads_dir}"/*3[80][804]*.fq
do
  echo "${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 \
--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 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}

# Generate FastA MD5 checksum
# See last line of SLURM output file
cd trinity_out_dir/
md5sum "${fasta_name}" > "${fasta_name}".checksum.md5

---

RESULTS

Remarkably quick; only ~1.5hrs:

Output folder:

• 20200527_cbai_trinity_arthropoda_pooled_RNAseq

Input FastQ list (text):

• fastq.list.txt

FastA (412MB):

• cbai_transcriptome_v1.7.fasta

  • MD5 = 032d1f81c7744736ebeefe7f63ed6d95

FastA Index (text):

• cbai_transcriptome_v1.7.fasta.fai

The following sets of files are useful for downstream gene expression and annotation using Trinity.

Trinity FastA Gene Trans Map (text):

• cbai_transcriptome_v1.7.fasta.gene_trans_map

Trinity FastA Sequence Lengths (text):

• cbai_transcriptome_v1.7.fasta.seq_lens

Assembly stats (text):

• assembly_stats.txt

################################
## Counts of transcripts, etc.
################################
Total trinity 'genes':	14225
Total trinity transcripts:	20526
Percent GC: 53.57

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

	Contig N10: 3760
	Contig N20: 2713
	Contig N30: 2122
	Contig N40: 1777
	Contig N50: 1504

	Median contig length: 777
	Average contig: 1053.71
	Total assembled bases: 21628372


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

	Contig N10: 3373
	Contig N20: 2449
	Contig N30: 1958
	Contig N40: 1645
	Contig N50: 1371

	Median contig length: 659
	Average contig: 930.99
	Total assembled bases: 13243346