I previously created a C.bairdi de novo transcriptome assembly with Trinity from the MEGAN6 taxonomic-specific reads for Arthropoda on 20200330 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):

20200407_cbai_busco_megan.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=cbai_busco_megan_transcriptome
## 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@uw.edu
## Specify the working directory for this job
#SBATCH --chdir=/gscratch/scrubbed/samwhite/outputs/20200407_cbai_busco_megan

# 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

# 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


# Establish variables for more readable code
timestamp=$(date +%Y%m%d)
species="cbai"
prefix="${timestamp}.${species}"

## Input files and settings
base_name="${prefix}.megan"
busco_db=/gscratch/srlab/sam/data/databases/BUSCO/metazoa_odb9
transcriptome_fasta=/gscratch/srlab/sam/data/C_bairdi/transcriptomes/20200406.C_bairdi.megan.Trinity.fasta
augustus_species=fly
threads=28

## Save working directory
wd=$(pwd)

## 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 ${base_name} \
--lineage_path ${busco_db} \
--mode transcriptome \
--cpu ${threads} \
--long \
--species ${augustus_species} \
--tarzip \
--augustus_parameters='--progress=true'

RESULTS

Very quick, 1.5 minutes:

Output folder:

20200407_cbai_busco_megan/

BUSCO short summary (text):

20200407_cbai_busco_megan/run_20200408.cbai.megan/short_summary_20200408.cbai.megan.txt

# BUSCO version is: 3.0.2
# The lineage dataset is: metazoa_odb9 (Creation date: 2016-02-13, number of species: 65, number of BUSCOs: 978)
# To reproduce this run: python /gscratch/srlab/programs/busco-v3/scripts/run_BUSCO.py -i /gscratch/srlab/sam/data/C_bairdi/transcriptomes/20200406.C_bairdi.megan.Trinity.fasta -o 20200407.cbai.megan -l /gscratch/srlab/sam/data/databases/BUSCO/metazoa_odb9/ -m transcriptome -c 28 --long -z
#
# Summarized benchmarking in BUSCO notation for file /gscratch/srlab/sam/data/C_bairdi/transcriptomes/20200406.C_bairdi.megan.Trinity.fasta
# BUSCO was run in mode: transcriptome

    C:91.8%[S:64.0%,D:27.8%],F:5.9%,M:2.3%,n:978

    898 Complete BUSCOs (C)
    626 Complete and single-copy BUSCOs (S)
    272 Complete and duplicated BUSCOs (D)
    58  Fragmented BUSCOs (F)
    22  Missing BUSCOs (M)
    978 Total BUSCO groups searched

In terms of BUSCO scoring, this iteration of the transcriptome is “better” (i.e. more complete) than the previous transcriptome BUSCO analysis on 20200207: 91.8% vs 85.5% complete BUSCOs identified. However, this is increase is primarily due to an increase in duplicated BUSCOs (27.8% vs 20.8%).