---
author: Sam White
toc-title: Contents
toc-depth: 5
toc-location: left
layout: post
title: Transcriptome Assessment - Crustacean Transcripome Completeness Evaluation Using BUSCO on Mox
date: '2020-11-10 11:10'
tags:
  - BUSCO
  - transcriptome
  - mox
categories:
  - 2020
  - Miscellaneous
---
Grace was recently working on writing up a manuscript which did a basic comparison of our _C.bairdi_ transcriptome (`cbai_transcriptome_v3.1`) (see the [Genomic Resources wiki](https://github.com/RobertsLab/resources/wiki/Genomic-Resources) for more deets) to two other species' transcriptome assemblies. We wanted BUSCO evaluations as part of this comparison, but the two other species did not have BUSCO scores in their respective publications. As such, I decided to generate them myself, as BUSCO runs very quickly. The job was run on Mox.

Info on the other two species' transcriptomes:

- _Carcinus maenas_ (green crab) transcriptome: [NCBI TSA](https://www.ncbi.nlm.nih.gov/nuccore/GBXE01000000)

  - Publication: [Verbruggen, Bas, Lisa K. Bickley, Eduarda M. Santos, Charles R. Tyler, Grant D. Stentiford, Kelly S. Bateman, and Ronny van Aerle. 2015. “De Novo Assembly of the Carcinus Maenas Transcriptome and Characterization of Innate Immune System Pathways.” BMC Genomics 16 (June): 458.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4469326/)

- _Litopenaeus vannamei_ (whiteleg shrimp) transcriptome: [OAKTrust](https://oaktrust.library.tamu.edu/handle/1969.1/152151)

  - Publication: [Novel transcriptome assembly and improved annotation of the whiteleg shrimp (Litopenaeus vannamei), a dominant crustacean in global seafood mariculture](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4243063/)



SBATCH script (GitHub):

- [20201110_crustacean-transcriptomes_busco.sh](https://github.com/RobertsLab/sams-notebook/blob/master/sbatch_scripts/20201110_crustacean-transcriptomes_busco.sh)

```shell
#!/bin/bash
## Job Name
#SBATCH --job-name=20201110_crustacean-transcriptomes_busco
## 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=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/20201110_crustacean-transcriptomes_busco
###################################################################################





# These variables need to be set by user

## Save working directory
wd=$(pwd)

# Transcriptomes array
transcriptomes_array=(
/gscratch/srlab/sam/data/C_maenas/transcriptomes/GBXE01.1.fsa_nt \
/gscratch/srlab/sam/data/L_vannamei/transcriptomes/Trinity_Trimmed_Whiteleg_Shrimp_Transcrimptome_Assmbled_Supplemental_Data_1.fasta
)



## Input files and settings
busco_db=/gscratch/srlab/sam/data/databases/BUSCO/metazoa_odb9
augustus_species=fly
threads=28

# Programs associative array
declare -A programs_array
programs_array=(
[busco]="/gscratch/srlab/programs/busco-v3/scripts/run_BUSCO.py"
)


## Set program paths
augustus_bin=/gscratch/srlab/programs/Augustus-3.3.2/bin
augustus_orig_config_dir=/gscratch/srlab/programs/Augustus-3.3.2/config
augustus_scripts=/gscratch/srlab/programs/Augustus-3.3.2/scripts
blast_dir=/gscratch/srlab/programs/ncbi-blast-2.8.1+/bin
hmm_dir=/gscratch/srlab/programs/hmmer-3.2.1/src

# Export Augustus variable
export PATH="${augustus_bin}:$PATH"
export PATH="${augustus_scripts}:$PATH"

## 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}"

# Copy BUSCO config file
cp ${busco_config_default} "${busco_config_ini}"

# 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}"

###################################################################################

# 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

for transcriptome in "${!transcriptomes_array[@]}"
do

  # Remove path from transcriptome using parameter substitution
  transcriptome_name="${transcriptomes_array[$transcriptome]##*/}"

  ## Augustus config directories
  augustus_dir=${wd}/${transcriptome_name}_augustus
  augustus_config_dir=${augustus_dir}/config


  export AUGUSTUS_CONFIG_PATH="${augustus_config_dir}"


  # 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}"


  # Run BUSCO/Augustus training
  ${programs_array[busco]} \
  --in ${transcriptomes_array[$transcriptome]} \
  --out ${transcriptome_name} \
  --lineage_path ${busco_db} \
  --mode transcriptome \
  --cpu ${threads} \
  --long \
  --species ${augustus_species} \
  --tarzip \
  --augustus_parameters='--progress=true'

  # Capture FastA checksums for verification
  echo ""
  echo "Generating checksum for ${transcriptome_name}"
  md5sum "${transcriptomes_array[$transcriptome]}" > "${transcriptome_name}".checksum.md5
  echo "Finished generating checksum for ${transcriptome_name}"
  echo ""

done


# 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

# Capture program options
for program in "${!programs_array[@]}"
do
	{
  echo "Program options for ${program}: "
	echo ""
	${programs_array[$program]} --help
	echo ""
	echo ""
	echo "----------------------------------------------"
	echo ""
	echo ""
} &>> program_options.log || true
done
```

---

# RESULTS

As usual, runtime was very quick ~8mins:

![Cumulative runtime for BUSCO evaluation of L.vannamei and C.maenas transcriptomes on Mox](https://github.com/RobertsLab/sams-notebook/blob/master/images/screencaps/20201110_crustacean-transcriptomes_busco_runtime.png?raw=true)

Output folder:

- [20201110_crustacean-transcriptomes_busco/](https://gannet.fish.washington.edu/Atumefaciens/20201110_crustacean-transcriptomes_busco/)

##### _C.maenas_

- [20201110_crustacean-transcriptomes_busco/run_GBXE01.1.fsa_nt/short_summary_GBXE01.1.fsa_nt.txt](https://gannet.fish.washington.edu/Atumefaciens/20201110_crustacean-transcriptomes_busco/run_GBXE01.1.fsa_nt/short_summary_GBXE01.1.fsa_nt.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_maenas/transcriptomes/GBXE01.1.fsa_nt -o GBXE01.1.fsa_nt -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_maenas/transcriptomes/GBXE01.1.fsa_nt
# BUSCO was run in mode: transcriptome

	C:95.7%[S:57.0%,D:38.7%],F:3.6%,M:0.7%,n:978

	935	Complete BUSCOs (C)
	557	Complete and single-copy BUSCOs (S)
	378	Complete and duplicated BUSCOs (D)
	35	Fragmented BUSCOs (F)
	8	Missing BUSCOs (M)
	978	Total BUSCO groups searched
```

##### _L.vannamei_

- [20201110_crustacean-transcriptomes_busco/run_Trinity_Trimmed_Whiteleg_Shrimp_Transcrimptome_Assmbled_Supplemental_Data_1.fasta/short_summary_Trinity_Trimmed_Whiteleg_Shrimp_Transcrimptome_Assmbled_Supplemental_Data_1.fasta.txt](https://gannet.fish.washington.edu/Atumefaciens/20201110_crustacean-transcriptomes_busco/run_Trinity_Trimmed_Whiteleg_Shrimp_Transcrimptome_Assmbled_Supplemental_Data_1.fasta/short_summary_Trinity_Trimmed_Whiteleg_Shrimp_Transcrimptome_Assmbled_Supplemental_Data_1.fasta.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/L_vannamei/transcriptomes/Trinity_Trimmed_Whiteleg_Shrimp_Transcrimptome_Assmbled_Supplemental_Data_1.fasta -o Trinity_Trimmed_Whiteleg_Shrimp_Transcrimptome_Assmbled_Supplemental_Data_1.fasta -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/L_vannamei/transcriptomes/Trinity_Trimmed_Whiteleg_Shrimp_Transcrimptome_Assmbled_Supplemental_Data_1.fasta
# BUSCO was run in mode: transcriptome

	C:98.1%[S:72.7%,D:25.4%],F:0.9%,M:1.0%,n:978

	959	Complete BUSCOs (C)
	711	Complete and single-copy BUSCOs (S)
	248	Complete and duplicated BUSCOs (D)
	9	Fragmented BUSCOs (F)
	10	Missing BUSCOs (M)
	978	Total BUSCO groups searched
```
