I previously created a C.bairdi de novo transcriptome assembly with Trinity from the MEGAN6 taxonomic-specific reads for Alveolata on 20200331 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):

20200331_hemat_busco_megan.sh

#!/bin/bash
## Job Name
#SBATCH --job-name=hemat_busco_megan_transcriptome
## Allocation Definition
#SBATCH --account=coenv
#SBATCH --partition=coenv
## Resources
## Nodes
#SBATCH --nodes=1
## Walltime (days-hours:minutes:seconds format)
#SBATCH --time=3-00:00:00
## 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/20200331_hemat_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=$(date +%Y%m%d)
species="hemat"
prefix="${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/Hematodinium/transcriptomes/20200408.hemat.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 fast! 49 seconds:

Output folder:

20200331_hemat_busco_megan

BUSCO short summary (text):

20200331_hemat_busco_megan/run_20200331.hemat.megan/short_summary_20200331.hemat.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/Hematodinium/transcriptomes/20200408.hemat.megan.Trinity.fasta -o 20200408.hemat.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/Hematodinium/transcriptomes/20200408.hemat.megan.Trinity.fasta
# BUSCO was run in mode: transcriptome

    C:25.6%[S:20.7%,D:4.9%],F:11.7%,M:62.7%,n:978

    250 Complete BUSCOs (C)
    202 Complete and single-copy BUSCOs (S)
    48  Complete and duplicated BUSCOs (D)
    114 Fragmented BUSCOs (F)
    614 Missing BUSCOs (M)
    978 Total BUSCO groups searched

This current assembly shows only a slight increase in completeness (25.6% complete BUSCOS), compared to the initial transcriptome assembly from 20200123 (25.1%).