pyprimer-ilps 0.1.0

A bioinformatic pipeline for identifying intron length variation across species to design diagnostic multiplex PCR primers.

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PyPrimer is bioinformatic pipeline designed to identify intron length variation between multiple species for the purpose of designing diagnostic multiplex PCR primers. Originally implemented in Neodiprion sawflies, the methodology can be applied to any system with a NCBI annotated reference genome.

PyPrimer was written in Python3.8+ and requires the following input files to function:

• table_OGs_protein_names.txt - Orthologous groups inferred using Broccoli (Derelle et al., 2020)
• GCF_021901455.1_iyNeoLeco1.1_feature_table.txt - NCBI feature tables in tab delimited format
• GCF_021901455.1_iyNeoLeco1.1_genomic.gff - NCBI GFF files

PyPrimer can optionally filter orthogroup candidates using genome wide FST population data for focal species. The original implementation focused on intron variation between N. lecontei and N. pinetum from Glover et al. (2024) using VCFtools.

PyPrimer can be run as a tutorial using files from the example_file directory on GitHub. Feature tables and GFF files will need to be downloaded from the NCBI ftp servers for each species.

Usage

# All genomes, no FST filter
pyprimer --genomes genomes.tsv \
         --broccoli-table table_OGs_protein_names.txt \
         --output-dir out/ \
         --min-intron-diff 100 --max-intron-len 1100

# Focal pair with FST filtering
pyprimer --genomes genomes.tsv \
         --broccoli-table table_OGs_protein_names.txt \
         --output-dir out/ \
         --use-fst --fst-table fst_file.txt --fst-cutoff 0.75 \
         --focal-pair pine leco \
         --min-intron-diff 100 --max-intron-len 1100

Mandatory arguments:
--genomes: path to the tab-seperate value file idetifying the number of species being considered, and the paths to their respecitive feature tables and .gff files.
--broccoli-table: path to the "table_OGs_protein_names.txt" file output by broccoli in the "dir_step3" directory.
--output-dir out: path to your desired output directory.
--min-intron-diff & --max-intron-lex: The minumum and maximum intron length desired between any two species.\

Optional Arguments:
--use-fst: Arugment to enable FST filtering.
--fst-table, --fst-cutoff: path to the file containing windowed FST values, along with the minimum desired FST threshold. Used in tandem with --use-fst.
--focal-pair: Argument to only search between two focal species.\

References

• Derelle, Romain, Hervé Philippe, and John K. Colbourne. "Broccoli: combining phylogenetic and network analyses for orthology assignment." Molecular Biology and Evolution 37.11 (2020): 3389-3396.

• Glover, Ashleigh N., et al. "Recurrent selection shapes the genomic landscape of differentiation between a pair of host‐specialized haplodiploids that diverged with gene flow" Molecular Ecology 33.18 (2024): e17509.