--- author: Sam White toc-title: Contents toc-depth: 5 toc-location: left title: Transcriptome Annotation - M.magister De Novo Transcriptome Assembly for DuMOAR Project Using TransDecoder on Mox date: '2023-11-07' draft: true engine: knitr categories: - mox - TransDecoder - annotation - transcriptome - 2023 - DuMOAR - Dungeness crab - Metacarcinus magister --- Began [transcriptome annotation](https://github.com/laurahspencer/DuMOAR/issues/44) (GitHub issue) using [`TransDecoder`](https://https://github.com/TransDecoder/TransDecoder/wiki.com/Trinotate/Trinotate/wiki) on the _de novo_ transcriptome assembly provide by Giles Goetz (see link to issue). [`TransDecoder`](https://https://github.com/TransDecoder/TransDecoder/wiki.com/Trinotate/Trinotate/wiki) uses a combination of [`BLASTp`](https://www.ncbi.nlm.nih.gov/books/NBK279690/) `hmmscan` and `pfam` to identify the longest potential open reading frames (ORFs) from the hundreds of thousands of contigs assembled by [`Trinity`](https://github.com/trinityrnaseq/trinityrnaseq/wiki). The job was run on Mox. # SLURM script - [20231107-mmag-transdecoder-transcriptome.sh](https://github.com/RobertsLab/sams-notebook/blob/master/sbatch_scripts/20231107-mmag-transdecoder-transcriptome.sh) (GitHub) ```bash #!/bin/bash ## Job Name #SBATCH --job-name=20231107-mmag-transdecoder-transcriptome ## Allocation Definition #SBATCH --account=srlab #SBATCH --partition=srlab ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=05-00:00:00 ## Memory per node #SBATCH --mem=200G ##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/20231107-mmag-transdecoder-transcriptome # Transdecoder to identify ORFs DuMOAR Trinity assembly ################################################################################### # These variables need to be set by user ## Assign Variables threads=28 # Paths to input/output files trinity_fasta="/gscratch/srlab/sam/data/C_magister/transcriptomes/trinity_denovo.Trinity.fasta" trinity_gene_trans_map="/gscratch/srlab/sam/data/C_magister/transcriptomes/trinity_denovo.Trinity.fasta.gene_trans_map" blastp_out_dir="blastp_out" transdecoder_out_dir="${trinity_fasta##*/}.transdecoder_dir" pfam_out_dir="pfam_out" blastp_out="${blastp_out_dir}/blastp.outfmt6" pfam_out="${pfam_out_dir}/pfam.domtblout" lORFs_pep="${transdecoder_out_dir}/longest_orfs.pep" pfam_db="/gscratch/srlab/programs/Trinotate-v3.1.1/admin/Pfam-A.hmm" sp_db="/gscratch/srlab/programs/Trinotate-v3.1.1/admin/uniprot_sprot.pep" # Paths to programs blast_dir="/gscratch/srlab/programs/ncbi-blast-2.8.1+/bin" blastp="${blast_dir}/blastp" hmmer_dir="/gscratch/srlab/programs/hmmer-3.2.1/src" hmmscan="${hmmer_dir}/hmmscan" transdecoder_dir="/gscratch/srlab/programs/TransDecoder-v5.5.0" transdecoder_lORFs="${transdecoder_dir}/TransDecoder.LongOrfs" transdecoder_predict="${transdecoder_dir}/TransDecoder.Predict" ################################################################################### # Load Python Mox module for Python module availability module load intel-python3_2017 # Capture input FastA checksum echo "Input Fasta: ${trinity_fasta}" echo "MD5 checksum:" echo "" md5sum "${trinity_fasta}" | tee --append md5checksum-assembly.txt echo "" # Make output directories mkdir --parents "${blastp_out_dir}" mkdir --parents "${pfam_out_dir}" # Extract long open reading frames ${transdecoder_lORFs} \ -t ${trinity_fasta} \ --gene_trans_map ${trinity_gene_trans_map} # Run blastp on long ORFs ${blastp} \ -query ${lORFs_pep} \ -db ${sp_db} \ -max_target_seqs 1 \ -outfmt 6 \ -evalue 1e-5 \ -num_threads ${threads} \ > ${blastp_out} # Run pfam search ${hmmscan} \ --cpu ${threads} \ --domtblout ${pfam_out} \ ${pfam_db} \ ${lORFs_pep} # Run Transdecoder with blastp and Pfam results ${transdecoder_predict} \ -t ${trinity_fasta} \ --retain_pfam_hits ${pfam_out} \ --retain_blastp_hits ${blastp_out} #################################################################### # Capture program options if [[ "${#programs_array[@]}" -gt 0 ]]; then echo "Logging program options..." for program in "${!programs_array[@]}" do { echo "Program options for ${program}: " echo "" # Handle samtools help menus if [[ "${program}" == "samtools_index" ]] \ || [[ "${program}" == "samtools_sort" ]] \ || [[ "${program}" == "samtools_view" ]] then ${programs_array[$program]} # Handle DIAMOND BLAST menu elif [[ "${program}" == "diamond" ]]; then ${programs_array[$program]} help # Handle NCBI BLASTx menu elif [[ "${program}" == "blastx" ]]; then ${programs_array[$program]} -help # Handle StringTie prepDE script elif [[ "${program}" == "prepDE" ]]; then python3 ${programs_array[$program]} -h fi ${programs_array[$program]} -h echo "" echo "" echo "----------------------------------------------" echo "" echo "" } &>> program_options.log || true # If MultiQC is in programs_array, copy the config file to this directory. if [[ "${program}" == "multiqc" ]]; then cp --preserve ~/.multiqc_config.yaml multiqc_config.yaml fi done echo "Finished logging programs options." echo "" fi # Document programs in PATH (primarily for program version ID) echo "Logging system $PATH..." { date echo "" echo "System PATH for $SLURM_JOB_ID" echo "" printf "%0.s-" {1..10} echo "${PATH}" | tr : \\n } >> system_path.log echo "Finished logging system $PATH." ``` /---------- # RESULTS ## Runtime Runtime was actually longer than I anticipated: ~1.25 days. ![TransDecoder runtime for M.magister transcriptome assembly of 1 day, 6hrs 49mins, and 28secs](20231107-mmag-transdecoder-transcriptome-runtime.png) ## Files Although there are other files generated by [`TransDecoder`](https://https://github.com/TransDecoder/TransDecoder/wiki.com/Trinotate/Trinotate/wiki), such as BEDs and GFFs, the following files are those utilized downstream by [`Trinotate`](https://github.com/Trinotate/Trinotate/wiki) to complete the transcriptome annotation pipeline. Output folder: - [20231107-mmag-transdecoder-transcriptome/](https://gannet.fish.washington.edu/Atumefaciens/20231107-mmag-transdecoder-transcriptome/) - FastA checksum (TXT): - [md5checksum-assembly.txt](https://gannet.fish.washington.edu/Atumefaciens/20231107-mmag-transdecoder-transcriptome/md5checksum-assembly.txt) - BLASTp output format 6 (TXT): - [20231107-mmag-transdecoder-transcriptome/blastp_out/blastp.outfmt6](https://gannet.fish.washington.edu/Atumefaciens/20231107-mmag-transdecoder-transcriptome/blastp_out/blastp.outfmt6) (6.5MB) - Longest peptide ORFs (FastA): - [20231107-mmag-transdecoder-transcriptome/trinity_denovo.Trinity.fasta.transdecoder_dir/longest_orfs.pep](https://gannet.fish.washington.edu/Atumefaciens/20231107-mmag-transdecoder-transcriptome/trinity_denovo.Trinity.fasta.transdecoder_dir/longest_orfs.pep) (57MB) - Pfam output (TXT): - [20231107-mmag-transdecoder-transcriptome/pfam_out/pfam.domtblout](https://gannet.fish.washington.edu/Atumefaciens/20231107-mmag-transdecoder-transcriptome/pfam_out/pfam.domtblout) (143MB)