Last updated: 2025-09-04

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Knit directory: locust-comparative-genomics/

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1. BUSCO assessment

To test whether core genes found in Insecta are present in the genomes that we will be analyzing, we run BUSCO to assess our Polyneoptera genome quality as recommended.

We ran in protein mode using the protein files XXX_proteins.faa downloaded from NCBI and stored in the folder 1_RawData using the following custom script for GRACE busco_assess.sh:

#!/bin/bash

#SBATCH --job-name=busco_all_proteins
#SBATCH --time=3-00:00:00
#SBATCH --cpus-per-task=48
#SBATCH --mem=250G
#SBATCH --ntasks=1
#SBATCH --output=busco_allproteins.%j.out
#SBATCH --error=busco_allproteins.%j.err

# Load required modules
module purge
ml GCC/12.2.0 OpenMPI/4.1.4 BUSCO/5.7.1 WebProxy

# Set environment variable for NumExpr to avoid "safe limit of 8"
export NUMEXPR_MAX_THREADS=$SLURM_CPUS_PER_TASK

# Set lineage and mode
LINEAGE=insecta_odb10
MODE=protein

# Set directories
INPUT_DIR=$PWD
OUTPUT_DIR=$PWD/busco_results
mkdir -p $OUTPUT_DIR

# Loop through all *_proteins.faa files
for FILE in ${INPUT_DIR}/*_proteins.faa; do
    BASENAME=$(basename "$FILE" _proteins.faa)
    echo "🔎 Starting BUSCO for: $BASENAME"

    busco -i "$FILE" \
          -o "${BASENAME}_busco" \
          -l "$LINEAGE" \
          -m "$MODE" \
          -c $SLURM_CPUS_PER_TASK \
          --out_path "$OUTPUT_DIR" \
          --force

    echo "✅ BUSCO run complete for $FILE"
done

echo "🎉 All BUSCO protein assessments completed!"

Just launch the script with:

#chmod 777 busco_assess.sh
sbatch busco_assess.sh

For 13 genomes, and using 48 CPUs, the run finished in ~5 hours and only used ~9 Gb of memory. We extracted the results from each species folder busco_results/{SPECIES}_busco/short_summary.specific.insecta_odb10.{SPECIES}_busco.txt.

2. Genome quality with Blobtools

We make snail plot with blobtools using the following commands. Unfortunately, because blobtools does not support indexing with .csi and our contigs are over 512 Mbp, we only plotted the GC, and length of our scaffolds and not the coverage.

First we

srun --ntasks 1 --cpus-per-task 24 --mem 50G --time 05:00:00 --pty bash

ml WebProxy 

# Check that the singularity container is properly installed
singularity exec /sw/hprc/sw/bio/containers/blobtoolkit/blobtoolkit_4.4.5.sif blobtools --help

# Create a BlobDir for each species
singularity exec /sw/hprc/sw/bio/containers/blobtoolkit/blobtoolkit_4.4.5.sif blobtools create --fasta /scratch/group/songlab/maeva/psmc-schistocerca/reference/cancellata.fasta BlobDir_cancellata

# Add mapping coverage using bwa mapping we 
# Does not plot on the snail plot
#singularity exec /sw/hprc/sw/bio/containers/blobtoolkit/blobtoolkit_4.4.5.sif blobtools add --cov ../results/bam/americana.sorted.bam BlobDir_americana

# Make a snail plot
singularity exec /sw/hprc/sw/bio/containers/blobtoolkit/blobtoolkit_4.4.5.sif blobtools view --view snail --plot BlobDir_cancellata

Example of snailplot obtained for S. cancellata



sessionInfo()
R version 4.4.2 (2024-10-31)
Platform: aarch64-apple-darwin20
Running under: macOS Sequoia 15.6.1

Matrix products: default
BLAS:   /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRblas.0.dylib 
LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.0

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

time zone: Asia/Tokyo
tzcode source: internal

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

loaded via a namespace (and not attached):
 [1] vctrs_0.6.5       cli_3.6.5         knitr_1.50        rlang_1.1.6      
 [5] xfun_0.52         stringi_1.8.7     promises_1.3.3    jsonlite_2.0.0   
 [9] workflowr_1.7.1   glue_1.8.0        rprojroot_2.0.4   git2r_0.36.2     
[13] htmltools_0.5.8.1 httpuv_1.6.16     sass_0.4.10       rmarkdown_2.29   
[17] tibble_3.3.0      evaluate_1.0.4    jquerylib_0.1.4   fastmap_1.2.0    
[21] yaml_2.3.10       lifecycle_1.0.4   whisker_0.4.1     stringr_1.5.1    
[25] compiler_4.4.2    fs_1.6.6          pkgconfig_2.0.3   Rcpp_1.0.14      
[29] rstudioapi_0.17.1 later_1.4.2       digest_0.6.37     R6_2.6.1         
[33] pillar_1.10.2     magrittr_2.0.3    bslib_0.9.0       tools_4.4.2      
[37] cachem_1.1.0