--- author: Sam White toc-title: Contents toc-depth: 5 toc-location: left layout: post title: Transcriptome Assembly - Geoduck Tissue-specific Assembly Ctenidia with HiSeq and NovaSeq Data on Mox date: '2019-04-09 06:44' tags: - Panopea generosa - geoduck - trinity - mox - assembly - transcriptome categories: - 2019 - Miscellaneous --- I previously assembled and annotated _P.generosa_ ctenidia transcriptome ([20190318](https://robertslab.github.io/sams-notebook/posts/2019/2019-03-18-Transcriptome-Annotation---Geoduck-Ctenidia-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_ctenidia_RNAseq.sh](https://github.com/RobertsLab/sams-notebook/blob/master/sbatch_scripts/20190409_trinity_pgen_ctenidia_RNAseq.sh) ```shell #!/bin/bash ## Job Name #SBATCH --job-name=trin_ctenidia ## 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_ctenidia_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 ${PATH} | tr : \\n >> system_path.log # User-defined variables reads_dir=/gscratch/scrubbed/samwhite/data/P_generosa/RNAseq/ctenidia threads=28 assembly_stats=assembly_stats.txt # Paths to programs trinity_dir="/gscratch/srlab/programs/Trinity-v2.8.3" samtools="/gscratch/srlab/programs/samtools-1.9/samtools" ## Inititalize arrays R1_array=() R2_array=() # Variables for R1/R2 lists R1_list="" R2_list="" # Create array of fastq R1 files R1_array=(${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 This took ~12hrs to complete. I'll pass this along to Steven/Christian, since this was done for Christian to use in some long, non-coding RNA (lncRNA) analysies. I'll also probably just take this through the annotation pipeline, since it's not difficult, nor time consuming. Output folder: - [20190409_trinity_pgen_ctenidia_RNAseq](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_ctenidia_RNAseq/) Trinity FastA: - 20190409_trinity_pgen_ctenidia_RNAseq/trinity_out_dir/Trinity.fasta[](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_ctenidia_RNAseq/trinity_out_dir/Trinity.fasta) Trinity FastA index file: - [20190409_trinity_pgen_ctenidia_RNAseq/trinity_out_dir/Trinity.fasta.fai](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_ctenidia_RNAseq/trinity_out_dir/Trinity.fasta.fai) Trinity Gene Trans Map file: - [20190409_trinity_pgen_ctenidia_RNAseq/trinity_out_dir/Trinity.fasta.gene_trans_map](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_ctenidia_RNAseq/trinity_out_dir/Trinity.fasta.gene_trans_map) Assembly stats (text): - [20190409_trinity_pgen_ctenidia_RNAseq/assembly_stats.txt](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_ctenidia_RNAseq/assembly_stats.txt) ``` ################################ ## Counts of transcripts, etc. ################################ Total trinity 'genes': 216248 Total trinity transcripts: 349773 Percent GC: 35.70 ######################################## Stats based on ALL transcript contigs: ######################################## Contig N10: 5199 Contig N20: 3549 Contig N30: 2617 Contig N40: 1927 Contig N50: 1387 Median contig length: 400 Average contig: 785.61 Total assembled bases: 274785010 ##################################################### ## Stats based on ONLY LONGEST ISOFORM per 'GENE': ##################################################### Contig N10: 4118 Contig N20: 2682 Contig N30: 1852 Contig N40: 1291 Contig N50: 892 Median contig length: 338 Average contig: 612.80 Total assembled bases: 132517038 ``` List of input FastQs (text): - [20190409_trinity_pgen_ctenidia_RNAseq/fastq.list.txt](http://gannet.fish.washington.edu/Atumefaciens/20190409_trinity_pgen_ctenidia_RNAseq/fastq.list.txt) ``` Geoduck-ctenidia-RNA-1_S3_L001_R1_001.fastq.gz Geoduck-ctenidia-RNA-1_S3_L001_R2_001.fastq.gz Geoduck-ctenidia-RNA-2_S11_L002_R1_001.fastq.gz Geoduck-ctenidia-RNA-2_S11_L002_R2_001.fastq.gz Geoduck-ctenidia-RNA-3_S19_L003_R1_001.fastq.gz Geoduck-ctenidia-RNA-3_S19_L003_R2_001.fastq.gz Geoduck-ctenidia-RNA-4_S27_L004_R1_001.fastq.gz Geoduck-ctenidia-RNA-4_S27_L004_R2_001.fastq.gz Geoduck-ctenidia-RNA-5_S35_L005_R1_001.fastq.gz Geoduck-ctenidia-RNA-5_S35_L005_R2_001.fastq.gz Geoduck-ctenidia-RNA-6_S43_L006_R1_001.fastq.gz Geoduck-ctenidia-RNA-6_S43_L006_R2_001.fastq.gz Geoduck-ctenidia-RNA-7_S51_L007_R1_001.fastq.gz Geoduck-ctenidia-RNA-7_S51_L007_R2_001.fastq.gz Geoduck-ctenidia-RNA-8_S59_L008_R1_001.fastq.gz Geoduck-ctenidia-RNA-8_S59_L008_R2_001.fastq.gz NR012_S1_L001_R1_001_val_1_val_1.fq.gz NR012_S1_L001_R2_001_val_2_val_2.fq.gz NR012_S1_L002_R1_001_val_1_val_1.fq.gz NR012_S1_L002_R2_001_val_2_val_2.fq.gz ```