After performing de novo assembly on our Tanner crab RNAseq data on 20191218 and performing BLASTx annotation on 20191224, I continued the annotation process by running Trinotate.

Trinotate will perform functional annotation of the transcriptome assembly, including GO terms and an annotation feature map that can be used in subsequent Trinity-based differential gene expression analysis so that functional annotations are carried downstream through that process.

SBATCH script (GitHub):

20191225_cbai_trinotate.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=trinotate_cbi
## Allocation Definition
#SBATCH --account=srlab
#SBATCH --partition=srlab
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=25-00:00:00
## 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 --chdir=/gscratch/scrubbed/samwhite/outputs/20191225_cbai_trinotate

# Exit script if any 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


wd="$(pwd)"
timestamp=$(date +%Y%m%d)
species="cbai"

prefix="${timestamp}.${species}.trinotate"


## Paths to input/output files

## New folders for working directory
rnammer_out_dir="${wd}/RNAmmer_out"
signalp_out_dir="${wd}/signalp_out"
tmhmm_out_dir="${wd}/tmhmm_out"

# Input files
blastp_out="/gscratch/scrubbed/samwhite/outputs/20191220_cbai_transdecoder/blastp_out/20191220.cbai.blastp.outfmt6"
blastx_out="/gscratch/scrubbed/samwhite/outputs/20191224_cbai_blastx_outfmt-11/20191224-20191218.C_bairdi.Trinity.fasta.blastx.outfmt6"
pfam_out="/gscratch/scrubbed/samwhite/outputs/20191220_cbai_transdecoder/pfam_out/20191220.cbai.pfam.domtblout"
lORFs_pep="/gscratch/scrubbed/samwhite/outputs/20191220_cbai_transdecoder/20191218.C_bairdi.Trinity.fasta.transdecoder_dir/longest_orfs.pep"
trinity_fasta="/gscratch/scrubbed/samwhite/outputs/20191218_cbai_trinity_RNAseq/trinity_out_dir/20191218.C_bairdi.Trinity.fasta"
trinity_gene_map="/gscratch/scrubbed/samwhite/outputs/20191218_cbai_trinity_RNAseq/trinity_out_dir/20191218.C_bairdi.Trinity.fasta.gene_trans_map"

# Output files
rnammer_out="${rnammer_out_dir}/20191218.C_bairdi.Trinity.fasta.rnammer.gff"
signalp_out="${signalp_out_dir}/${prefix}.signalp.out"
tmhmm_out="${tmhmm_out_dir}/${prefix}.tmhmm.out"
trinotate_report="${wd}/${prefix}_annotation_report.txt"

# Paths to programs
rnammer_dir="/gscratch/srlab/programs/RNAMMER-1.2"
rnammer="${rnammer_dir}/rnammer"
signalp_dir="/gscratch/srlab/programs/signalp-4.1"
signalp="${signalp_dir}/signalp"
tmhmm_dir="/gscratch/srlab/programs/tmhmm-2.0c/bin"
tmhmm="${tmhmm_dir}/tmhmm"
trinotate_dir="/gscratch/srlab/programs/Trinotate-v3.1.1"
trinotate="${trinotate_dir}/Trinotate"
trinotate_rnammer="${trinotate_dir}/util/rnammer_support/RnammerTranscriptome.pl"
trinotate_GO="${trinotate_dir}/util/extract_GO_assignments_from_Trinotate_xls.pl"
trinotate_features="${trinotate_dir}/util/Trinotate_get_feature_name_encoding_attributes.pl"
trinotate_sqlite_db="Trinotate.sqlite"

# Make output directories
mkdir "${rnammer_out_dir}" "${signalp_out_dir}" "${tmhmm_out_dir}"

# Copy sqlite database template

cp ${trinotate_dir}/admin/Trinotate.sqlite .

# Run signalp
${signalp} \
-f short \
-n "${signalp_out}" \
${lORFs_pep}

# Run tmHMM
${tmhmm} \
--short \
< ${lORFs_pep} \
> "${tmhmm_out}"

# Run RNAmmer
cd "${rnammer_out_dir}" || exit
${trinotate_rnammer} \
--transcriptome ${trinity_fasta} \
--path_to_rnammer ${rnammer}
cd "${wd}" || exit

# Run Trinotate
## Load transcripts and coding regions into database
${trinotate} \
${trinotate_sqlite_db} \
init \
--gene_trans_map "${trinity_gene_map}" \
--transcript_fasta "${trinity_fasta}" \
--transdecoder_pep "${lORFs_pep}"

## Load BLAST homologies
"${trinotate}" \
"${trinotate_sqlite_db}" \
LOAD_swissprot_blastp \
"${blastp_out}"

"${trinotate}" \
"${trinotate_sqlite_db}" \
LOAD_swissprot_blastx \
"${blastx_out}"

## Load Pfam
"${trinotate}" \
"${trinotate_sqlite_db}" \
LOAD_pfam \
"${pfam_out}"

## Load transmembrane domains
"${trinotate}" \
"${trinotate_sqlite_db}" \
LOAD_tmhmm \
"${tmhmm_out}"

## Load signal peptides
"${trinotate}" \
"${trinotate_sqlite_db}" \
LOAD_signalp \
"${signalp_out}"

## Load RNAmmer
"${trinotate}" \
"${trinotate_sqlite_db}" \
LOAD_rnammer \
"${rnammer_out}"

## Creat annotation report
"${trinotate}" \
"${trinotate_sqlite_db}" \
report \
> "${trinotate_report}"

# Extract GO terms from annotation report
"${trinotate_GO}" \
--Trinotate_xls "${trinotate_report}" \
-G \
--include_ancestral_terms \
> "${prefix}".go_annotations.txt

# Make transcript features annotation map
"${trinotate_features}" \
"${trinotate_report}" \
> "${prefix}".annotation_feature_map.txt

RESULTS

This took ~6hr to run (which was faster than I expected):

Output folder:

20191225_cbai_trinotate/

Annotation feature map. This can be used to update Trinity-based gene expression matrices like so:

${TRINITY_HOME}/Analysis/DifferentialExpression/rename_matrix_feature_identifiers.pl

20200104.cbai.trinotate.annotation_feature_map.txt

Annotation report (CSV)

20200104.cbai.trinotate_annotation_report.txt

Gene ontology (GO) annotations (TXT)

20200104.cbai.trinotate.go_annotations.txt

SQlite database:

Trinotate.sqlite

It should be remembered that the assembly was generated from infected/uninfected crabs, so there will be crab and Hematodinium sequences present.