--- author: Sam White toc-title: Contents toc-depth: 5 toc-location: left layout: post title: Transcriptome Assembly - Geoduck Tissue-specific Assembly Gonad HiSeq and NovaSeq Data on Mox date: '2019-04-09 07:00' tags: - Panopea generosa - geoduck - gonad - trinity - assembly - transcriptome - mox categories: - 2019 - Miscellaneous --- I previously assembled and annotated _P.generosa_ gonad transcriptome ([20190318](https://robertslab.github.io/sams-notebook/posts/2019/2019-03-18-Transcriptome-Annotation---Geoduck-Gonad-with-Trinotate-on-Mox/)) using just our HiSeq data from our Illumina collaboration. This was a an oversight, as I didn't realize that we also had NovaSeq RNAseq data. So, I've initiated another _de novo_ assembly using Trinity incorporating both sets of data. NovaSeq data had been [previously trimmed](https://robertslab.github.io/sams-notebook/posts/2018/2018-01-25-adapter-trimming-and-fastqc-illumina-geoduck-novaseq-data/). Trimming of the HiSeq data was performed via Trinity, using the `--trimmomatic` option. SBATCH script (GitHub): - [20190409_trinity_pgen_gonad_RNAseq.sh](https://github.com/RobertsLab/sams-notebook/blob/master/sbatch_scripts/20190409_trinity_pgen_gonad_RNAseq.sh) ```shell #!/bin/bash ## Job Name #SBATCH --job-name=trin_gonad ## 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_gonad_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 ${PATH} | tr : \\n >> system_path.log # User-defined variables reads_dir=/gscratch/scrubbed/samwhite/data/P_generosa/RNAseq/gonad threads=28 assembly_stats=assembly_stats.txt # Paths to programs trinity_dir="/gscratch/srlab/programs/Trinity-v2.8.3" samtools="/gscratch/srlab/programs/samtools-1.9/samtools" ## Inititalize arrays R1_array=() R2_array=() # Variables for R1/R2 lists R1_list="" R2_list="" # Create array of fastq R1 files R1_array=(${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: - [20190409_trinity_pgen_gonad_RNAseq/](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_gonad_RNAseq/) Trinity FastA: - [20190409_trinity_pgen_gonad_RNAseq/trinity_out_dir/Trinity.fasta](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_gonad_RNAseq/trinity_out_dir/Trinity.fasta) Trinity FastA index file: - [20190409_trinity_pgen_gonad_RNAseq/trinity_out_dir/Trinity.fasta.fai](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_gonad_RNAseq/trinity_out_dir/Trinity.fasta.fai) Trinity Gene Trans Map file: - [20190409_trinity_pgen_gonad_RNAseq/trinity_out_dir/Trinity.fasta.gene_trans_map](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_gonad_RNAseq/trinity_out_dir/Trinity.fasta.gene_trans_map) Assembly stats (text): - [20190409_trinity_pgen_gonad_RNAseq/assembly_stats.txt](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_gonad_RNAseq/assembly_stats.txt) ``` ################################ ## Counts of transcripts, etc. ################################ Total trinity 'genes': 151263 Total trinity transcripts: 198748 Percent GC: 36.21 ######################################## Stats based on ALL transcript contigs: ######################################## Contig N10: 3270 Contig N20: 2036 Contig N30: 1343 Contig N40: 908 Contig N50: 640 Median contig length: 315 Average contig: 522.32 Total assembled bases: 103810995 ##################################################### ## Stats based on ONLY LONGEST ISOFORM per 'GENE': ##################################################### Contig N10: 2267 Contig N20: 1319 Contig N30: 873 Contig N40: 626 Contig N50: 472 Median contig length: 288 Average contig: 439.32 Total assembled bases: 66453310 ``` List of input FastQs (text): - [20190409_trinity_pgen_gonad_RNAseq/fastq.list.txt](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_gonad_RNAseq/fastq.list.txt) ``` Geoduck-gonad-RNA-1_S1_L001_R1_001.fastq.gz Geoduck-gonad-RNA-1_S1_L001_R2_001.fastq.gz Geoduck-gonad-RNA-2_S9_L002_R1_001.fastq.gz Geoduck-gonad-RNA-2_S9_L002_R2_001.fastq.gz Geoduck-gonad-RNA-3_S17_L003_R1_001.fastq.gz Geoduck-gonad-RNA-3_S17_L003_R2_001.fastq.gz Geoduck-gonad-RNA-4_S25_L004_R1_001.fastq.gz Geoduck-gonad-RNA-4_S25_L004_R2_001.fastq.gz Geoduck-gonad-RNA-5_S33_L005_R1_001.fastq.gz Geoduck-gonad-RNA-5_S33_L005_R2_001.fastq.gz Geoduck-gonad-RNA-6_S41_L006_R1_001.fastq.gz Geoduck-gonad-RNA-6_S41_L006_R2_001.fastq.gz Geoduck-gonad-RNA-7_S49_L007_R1_001.fastq.gz Geoduck-gonad-RNA-7_S49_L007_R2_001.fastq.gz Geoduck-gonad-RNA-8_S57_L008_R1_001.fastq.gz Geoduck-gonad-RNA-8_S57_L008_R2_001.fastq.gz NR006_S3_L001_R1_001_val_1_val_1.fq.gz NR006_S3_L001_R2_001_val_2_val_2.fq.gz NR006_S3_L002_R1_001_val_1_val_1.fq.gz NR006_S3_L002_R2_001_val_2_val_2.fq.gz ```