Transcriptome Annotation – DIAMOND BLASTx on C.bairdi Transcriptome v4.0 on Mox

Continued annotation of cbai_transcriptome_v4.0.fasta [Trinity de novo assembly from 20210317(https://ift.tt/2NziJW6] using DIAMOND BLASTx on Mox. This will be used as a component of Trinotate annotation downstream.

SBATCH script (GitHub):

• 20210318_cbai_diamond_blastx_transcriptome-v4.0.sh

#!/bin/bash ## Job Name #SBATCH --job-name=20210318_cbai_diamond_blastx_transcriptome-v4.0 ## Allocation Definition #SBATCH --account=coenv #SBATCH --partition=coenv ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=10-00:00:00 ## Memory per node #SBATCH --mem=120G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20210318_cbai_diamond_blastx_transcriptome-v4.0 ### BLASTx of Trinity de novo assembly of all C.bairdi RNAseq with BLASTx matches to C.opilio genome. ### cbai_transcriptome_v4.0.fasta ### Includes RNAseq short-hand of: 2020-GW, 2020-UW, 2019, 2018. ################################################################################### # These variables need to be set by user # Exit script if any command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # SegFault fix? export THREADS_DAEMON_MODEL=1 # Programs array declare -A programs_array programs_array=( [diamond]="/gscratch/srlab/programs/diamond-0.9.29/diamond" ) # DIAMOND UniProt database dmnd=/gscratch/srlab/blastdbs/uniprot_sprot_20200123/uniprot_sprot.dmnd # Trinity assembly (FastA) fasta=/gscratch/srlab/sam/data/C_bairdi/transcriptomes/cbai_transcriptome_v4.0.fasta ################################################################################### # Strip leading path and extensions no_path=$(echo "${fasta##*/}") no_ext=$(echo "${no_path%.*}") # Run DIAMOND with blastx # Output format 6 produces a standard BLAST tab-delimited file ${programs_array[diamond]} blastx \ --db ${dmnd} \ --query "${fasta}" \ --out "${no_ext}".blastx.outfmt6 \ --outfmt 6 \ --evalue 1e-4 \ --max-target-seqs 1 \ --block-size 15.0 \ --index-chunks 4 # Generate checksums for future reference echo "" echo "Generating checksum for ${fasta}." md5sum "${fasta}">> fastq.checksums.md5 echo "Completed checksum for ${fasta}." echo "" ################################################################################### # Capture program options echo "Logging program options..." for program in "${!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 fi ${programs_array[$program]} -h echo "" echo "" echo "

TransDecoder – C.bairdi Transcriptome v4.0 on Mox

Began annotation of cbai_transcriptome_v4.0.fasta [Trinity de novo assembly from 20210317(https://ift.tt/2NziJW6] using TransDecoder on Mox. This will be used as a component of Trinotate annotation downstream.

SBATCH script (GitHub):

• 20210317_cbai_transdecoder_transcriptome_v4.0.sh

#!/bin/bash ## Job Name #SBATCH --job-name=20210317_cbai_transdecoder_transcriptome_v4.0 ## Allocation Definition #SBATCH --account=coenv #SBATCH --partition=coenv ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=8-00:00:00 ## Memory per node #SBATCH --mem=120G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20210317_cbai_transdecoder_transcriptome_v4.0 # Exit script if a command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # Set workind directory as current directory wd="$(pwd)" # Set input file locations trinity_fasta="/gscratch/srlab/sam/data/C_bairdi/transcriptomes/cbai_transcriptome_v4.0.fasta" trinity_gene_map="/gscratch/srlab/sam/data/C_bairdi/transcriptomes/cbai_transcriptome_v4.0.fasta.gene_trans_map" # Capture trinity file name trinity_fasta_name=${trinity_fasta##*/} # Paths to input/output files blastp_out_dir="${wd}/blastp_out" transdecoder_out_dir="${wd}/${trinity_fasta_name}.transdecoder_dir" pfam_out_dir="${wd}/pfam_out" blastp_out="${blastp_out_dir}/${trinity_fasta_name}.blastp.outfmt6" pfam_out="${pfam_out_dir}/${trinity_fasta_name}.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="${transdecoder_dir}/TransDecoder.Predict" # Capture FastA MD5 checksum for future reference md5sum "${trinity_fasta}" >> "${trinity_fasta_name}".checksum.md5 # Make output directories mkdir "${blastp_out_dir}" mkdir "${pfam_out_dir}" # Extract long open reading frames "${transdecoder_lORFs}" \ --gene_trans_map "${trinity_gene_map}" \ -t "${trinity_fasta}" # Run blastp on long ORFs "${blastp}" \ -query "${lORFs_pep}" \ -db "${sp_db}" \ -max_target_seqs 1 \ -outfmt 6 \ -evalue 1e-5 \ -num_threads 28 \ > "${blastp_out}" # Run pfam search "${hmmscan}" \ --cpu 28 \ --domtblout "${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}" # 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 

Transcriptome Assessment – BUSCO Metazoa on C.bairdi Transcriptome v4.0 on Mox

I previously created a C.bairdi de novo transcriptome assembly v4.0 with Trinity from all our C.bairdi RNAseq reads which had BLASTx matches to the C.opilio genome and decided to assess its “completeness” using BUSCO and the metazoa_odb9 database.

BUSCO was run with the --mode transcriptome option on Mox.

SBATCH script (GitHub):

• 20210317_cbai_busco_transcriptome_v4.0.sh

#!/bin/bash ## Job Name #SBATCH --job-name=20210317_cbai_busco_transcriptome_v4.0 ## Allocation Definition #SBATCH --account=coenv #SBATCH --partition=coenv ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=1-00:00:00 ## Memory per node #SBATCH --mem=120G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20210317_cbai_busco_transcriptome_v4.0 ### C.bairdi transcriptome assembly completeness assessment using BUSCO. ### This is checking cbai_transcriptome_v4.0 fasta # Load Python Mox module for Python module availability module load intel-python3_2017 # Load Open MPI module for parallel, multi-node processing module load icc_19-ompi_3.1.2 # SegFault fix? export THREADS_DAEMON_MODEL=1 ## Input files and settings busco_db=/gscratch/srlab/sam/data/databases/BUSCO/metazoa_odb9 transcriptome_fasta=/gscratch/srlab/sam/data/C_bairdi/transcriptomes/cbai_transcriptome_v4.0.fasta augustus_species=fly threads=40 ## Save working directory wd=$(pwd) # Extract FastA filename fasta_name=${transcriptome_fasta##*/} ## Set program paths augustus_bin=/gscratch/srlab/programs/Augustus-3.3.2/bin augustus_scripts=/gscratch/srlab/programs/Augustus-3.3.2/scripts blast_dir=/gscratch/srlab/programs/ncbi-blast-2.8.1+/bin/ busco=/gscratch/srlab/programs/busco-v3/scripts/run_BUSCO.py hmm_dir=/gscratch/srlab/programs/hmmer-3.2.1/src/ ## Augustus configs augustus_dir=${wd}/augustus augustus_config_dir=${augustus_dir}/config augustus_orig_config_dir=/gscratch/srlab/programs/Augustus-3.3.2/config ## BUSCO configs busco_config_default=/gscratch/srlab/programs/busco-v3/config/config.ini.default busco_config_ini=${wd}/config.ini # Export BUSCO config file location export BUSCO_CONFIG_FILE="${busco_config_ini}" # Export Augustus variable export PATH="${augustus_bin}:$PATH" export PATH="${augustus_scripts}:$PATH" export AUGUSTUS_CONFIG_PATH="${augustus_config_dir}" # Copy BUSCO config file cp ${busco_config_default} "${busco_config_ini}" # Make Augustus directory if it doesn't exist if [ ! -d "${augustus_dir}" ]; then mkdir --parents "${augustus_dir}" fi # Copy Augustus config directory cp --preserve -r ${augustus_orig_config_dir} "${augustus_dir}" # Edit BUSCO config file ## Set paths to various programs ### The use of the % symbol sets the delimiter sed uses for arguments. ### Normally, the delimiter that most examples use is a slash "/". ### But, we need to expand the variables into a full path with slashes, which screws up sed. ### Thus, the use of % symbol instead (it could be any character that is NOT present in the expanded variable; doesn't have to be "%"). sed -i "/^;cpu/ s/1/${threads}/" "${busco_config_ini}" sed -i "/^tblastn_path/ s%tblastn_path = /usr/bin/%path = ${blast_dir}%" "${busco_config_ini}" sed -i "/^makeblastdb_path/ s%makeblastdb_path = /usr/bin/%path = ${blast_dir}%" "${busco_config_ini}" sed -i "/^augustus_path/ s%augustus_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/bin/%path = ${augustus_bin}%" "${busco_config_ini}" sed -i "/^etraining_path/ s%etraining_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/bin/%path = ${augustus_bin}%" "${busco_config_ini}" sed -i "/^gff2gbSmallDNA_path/ s%gff2gbSmallDNA_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/scripts/%path = ${augustus_scripts}%" "${busco_config_ini}" sed -i "/^new_species_path/ s%new_species_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/scripts/%path = ${augustus_scripts}%" "${busco_config_ini}" sed -i "/^optimize_augustus_path/ s%optimize_augustus_path = /home/osboxes/BUSCOVM/augustus/augustus-3.2.2/scripts/%path = ${augustus_scripts}%" "${busco_config_ini}" sed -i "/^hmmsearch_path/ s%hmmsearch_path = /home/osboxes/BUSCOVM/hmmer/hmmer-3.1b2-linux-intel-ia32/binaries/%path = ${hmm_dir}%" "${busco_config_ini}" # Run BUSCO/Augustus training ${busco} \ --in ${transcriptome_fasta} \ --out ${fasta_name} \ --lineage_path ${busco_db} \ --mode transcriptome \ --cpu ${threads} \ --long \ --species ${augustus_species} \ --tarzip \ --augustus_parameters='--progress=true' # Create checksum for potential verification md5sum "${transcriptome_fasta}" >> "${fasta_name}".checksum.md5 

Transcriptome Assembly – C.bairdi Transcriptome v4.0 Using Trinity on Mox

Continuing to addressing this GitHub issue, to generate an additional C.bairdi transcriptome, I finally got to the point of actually running the assembly using Trinity using the extracted reads from 20210316. Those reads were identified via BLASTx agianst the C.opilio genome proteins on 20210312. Trinty was run on Mox.

SBATCH script (GitHub):

• 20210317_cbai_trinity_RNAseq_transcriptome-v4.0.sh

#!/bin/bash ## Job Name #SBATCH --job-name=20210317_cbai_trinity_RNAseq_transcriptome-v4.0 ## Allocation Definition #SBATCH --account=coenv #SBATCH --partition=coenv ## Resources ## Nodes #SBATCH --nodes=1 ## Walltime (days-hours:minutes:seconds format) #SBATCH --time=20-00:00:00 ## Memory per node #SBATCH --mem=200G ##turn on e-mail notification #SBATCH --mail-type=ALL #SBATCH --mail-user=samwhite ## Specify the working directory for this job #SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20210317_cbai_trinity_RNAseq_transcriptome-v4.0 ## Trinity assembly of C.bairdi RNAseq reads ## with BLASTx matches to NCBI C.opilio genome proteins (GCA_016584305.1) ## Assembly will be referred to as cbai_transcriptome_v4.0 ################################################################################### # These variables need to be set by user # Path to this script script_path=/gscratch/scrubbed/samwhite/outputs/20210317_cbai_trinity_RNAseq_transcriptome-v4.0/20210317_cbai_trinity_RNAseq_transcriptome-v4.0.sh # RNAseq FastQs directory reads_dir=/gscratch/scrubbed/samwhite/outputs/20210316_cbai-vs-copi_reads_extractions # Transcriptomes directory transcriptomes_dir=/gscratch/srlab/sam/data/C_bairdi/transcriptomes # CPU threads threads=40 # Capture specified RAM from this script # Carrot needed to limit grep to line starting with #SBATCH # Avoids grep-ing the command below. max_mem=$(grep "^#SBATCH --mem=" ${script_path} | awk -F [=] '{print $2}') # Paths to programs trinity_dir="/gscratch/srlab/programs/trinityrnaseq-v2.12.0" samtools="/gscratch/srlab/programs/samtools-1.10/samtools" # Set transcriptome name transcriptome_name="cbai_transcriptome_v4.0.fasta" # Programs array declare -A programs_array programs_array=( [samtools_faidx]="${samtools} faidx" \ [trinity]="${trinity_dir}/Trinity" \ [trinity_stats]="${trinity_dir}/util/TrinityStats.pl" \ [trinity_gene_trans_map]="${trinity_dir}/util/support_scripts/get_Trinity_gene_to_trans_map.pl" \ [trinity_fasta_seq_length]="${trinity_dir}/util/misc/fasta_seq_length.pl" ) # FastQ array fastq_array=(${reads_dir}/*copi-BLASTx-match.fq.gz) # Variables R1_list="" R2_list="" ################################################################################### # Exit script if any command fails set -e # Load Python Mox module for Python module availability module load intel-python3_2017 # Set variables trinity_out_dir="" assembly_stats="${transcriptome_name}_assembly_stats.txt" trinity_out_dir="${transcriptome_name}_trinity_out_dir" # Adds empty line between checksums and next info logged to SLURM output. echo "" # Create comma-separated lists of FastQ reads # Loop through read pairs # Increment by 2 to process next pair of FastQ files for (( i=0; i<${#fastq_array[@]} ; i+=2 )) do # Handle "fence post" problem # associated with comma placement if [[ ${i} -eq 0 ]]; then R1_list="${fastq_array[${i}]}," R2_list="${fastq_array[${i}+1]}," elif [[ ${i} -eq $(( ${#fastq_array[@]} - 1 )) ]]; then R1_list="${R1_list}${fastq_array[${i}]}" R2_list="${R2_list}${fastq_array[${i}+1]}" else R1_list="${R1_list}${fastq_array[${i}]}," R2_list="${R2_list}${fastq_array[${i}+1]}," fi done # Run Trinity without stranded RNAseq option ${programs_array[trinity]} \ --seqType fq \ --max_memory ${max_mem} \ --CPU ${threads} \ --output ${trinity_out_dir} \ --left "${R1_list}" \ --right "${R2_list}" # Rename generic assembly FastA mv "${trinity_out_dir}"/Trinity.fasta "${trinity_out_dir}"/"${transcriptome_name}" # Assembly stats ${programs_array[trinity_stats]} "${trinity_out_dir}"/"${transcriptome_name}" \ > "${assembly_stats}" # Create gene map files ${programs_array[trinity_gene_trans_map]} \ "${trinity_out_dir}"/"${transcriptome_name}" \ > "${trinity_out_dir}"/"${transcriptome_name}".gene_trans_map # Create sequence lengths file (used for differential gene expression) ${programs_array[trinity_fasta_seq_length]} \ "${trinity_out_dir}"/"${transcriptome_name}" \ > "${trinity_out_dir}"/"${transcriptome_name}".seq_lens # Create FastA index ${programs_array[samtools_faidx]} \ "${trinity_out_dir}"/"${transcriptome_name}" # Copy files to transcriptomes directory rsync -av \ "${trinity_out_dir}"/"${transcriptome_name}"* \ ${transcriptomes_dir} # Capture FastA checksums for verification cd "${trinity_out_dir}"/ echo "" echo "Generating checksum for ${transcriptome_name}" md5sum "${transcriptome_name}" > "${transcriptome_name}".checksum.md5 echo "Finished generating checksum for ${transcriptome_name}" echo "" # Generate input FastQ checksums for fastq in "${!fastq_array[@]}" do echo "" echo "Generating checksum for ${fastq_array[$fastq]}" md5sum "${fastq_array[$fastq]}" >> fastq_checksums.md5 echo "Checksum for ${fastq_array[$fastq]} complete." done ################################################################################### # Capture program options 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 fi ${programs_array[$program]} -h echo "" echo "" echo "