================================================================================Genomics and Machine Learning Uncover Diapause Mechanisms and Predict Voltinism in the Bark Beetle Ips typographus================================================================================ DATASET DESCRIPTION-------------------This repository contains the code and analytical pipeline used to identify genetic polymorphisms associated with diapause phenotypes in the European spruce bark beetle, Ips typographus. The analysis combines genome-wide association study (GWAS) with machine learning (ML) to predict facultative versus obligate diapause phenotypes, with implications for understanding voltinism and outbreak risk. ================================================================================STUDY OVERVIEW================================================================================ Citation: Palmieri, L., Dowle, E.J., Nadachowska-Brzyska, K., Schopf, A., Dobart, N., Stauffer, C., Schuler, H., Ragland, G.J., & Schebeck, M. (2025). Genomics and Machine Learning Uncover Diapause Mechanisms and Predict Voltinism in the Bark Beetle Ips typographus. Molecular Ecology. Authors: Luciano Palmieri Contact: luciano.palmierirocha@unibz.it code revised with Claude Sonnet 4.5 ================================================================================ABBREVIATIONS AND CODES================================================================================ Sample Population Codes------------------------* LOW: Central European low-altitude population (Prinzersdorf, Austria; 48°22'N, 15°48'E; 350 m)* HIGH: Central European high-altitude population (Gesäuse, Austria; 47°35'N, 14°38'E; 1500 m)* NORTH: Northern European population (Vindeln, Sweden; 64°12'N, 19°43'E; 300 m) Phenotype Classifications-------------------------* Facultative diapause: Individuals capable of producing multiple generations per year under favorable conditions* Obligate diapause: Individuals with univoltine life cycle regardless of environmental conditions Technical Abbreviations-----------------------* GWAS: Genome-Wide Association Study* ML: Machine Learning* BF: Bayes Factor (strength of association in GWAS)* VCA: Variance Component Analysis* VC: Variance Component* SNP: Single Nucleotide Polymorphism* ddRAD: Double-Digest Restriction-Associated DNA sequencing* AUROC: Area Under the Receiver Operating Characteristic Curve* GO: Gene Ontology* JHE: Juvenile Hormone Esterase* LG: Linkage Group (chromosome) ================================================================================FILE DESCRIPTIONS================================================================================ 1. params-Ips_ipyrad.txt-------------------------Description: Parameter file for ipyrad v.0.9.95 assemblyPurpose: Configuration for ddRAD sequence assembly and SNP callingUsage: Input for ipyrad pipeline to generate VCF files from raw sequencing data 2. phenotype_pruned_filtered75missing_LD.vcf---------------------------------------------Description: Filtered and LD-pruned VCF file for phenotyped individualsPurpose: Final quality-controlled SNP dataset for GWAS and ML analysesContent: 11,867 SNPs across 296 phenotyped individuals after filtering for: - Maximum 25% missing data per SNP - Linkage disequilibrium (r² 6) and ML analysis - Gene annotation file (GFF/GTF) - Gene-to-GO mapping file - Gene length fileOutput: - SNP-to-gene mappings - GO enrichment results - Enriched biological pathwaysDependencies: GenomicRanges, goseq ================================================================================ANALYTICAL PIPELINE (ORDER OF EXECUTION)================================================================================ STEP 1: RAD Sequencing and SNP Calling---------------------------------------Command: ipyrad -p params-Ips_ipyrad.txt -s 1234567Input: Raw ddRAD sequencing reads (available at NCBI SRA: PRJDB38037)Output: VCF file with ~134,410 SNPs across 574 individuals STEP 2: Population Structure Analysis--------------------------------------Command: Rscript VCA_and_PopGEN_script.rInput: Raw VCF file from Step 1Output: VC scores, population structure plots STEP 3: VCF Filtering, Gene Annotation, and GWAS-------------------------------------------------Command (Windows): Filtering_Liftoff_and_Baypass_GWAS.batCommand (Linux/Mac): bash Filtering_Liftoff_and_Baypass_GWAS.txt Input: - Filtered VCF from ipyrad - Reference genome and annotations - VC scores from Step 2 Output: - Filtered VCF (11,867 SNPs after QC and LD pruning) - Gene annotations - Baypass results with Bayes Factors for each SNP Key GWAS Results: - 185 SNPs with BF > 2 (weak association) - 48 SNPs with BF > 6 (moderate association) - 13 SNPs with BF > 10 (strong association) - Top SNP: Juvenile hormone esterase (JHE) locus (BF = 18.42) STEP 4: Machine Learning - Hyperparameter Optimization-------------------------------------------------------Command: python Hyperparameter_tuning.pyInput: Phenotyped individuals SNP matrix + VC scoresOutput: Optimal hyperparameters (learning rate, number of leaves, depth, etc.) STEP 5: Machine Learning - Model Training and Validation---------------------------------------------------------Command: python LightGBM_training_KFold.py Input: - Phenotyped individuals (296 samples) - Top 25 ML-ranked SNPs + GWAS SNPs (BF > 6) - VC1, VC2, VC3 scores - Optimized hyperparameters Output: - Trained model: BEST_model_phenotyped_kfold_lgbmAUROC.pkl - SNP importance rankings - Performance: AUROC = 0.918, Mean Accuracy = 0.856 Top ML Results: - JHE locus ranked #1 (importance score = 2847.81) - Only 2 SNPs overlapped between GWAS BF>10 and ML top 25 - Best model: ML top 25 + GWAS BF>6 + VCA STEP 6: Phenotype Prediction in Wild Populations-------------------------------------------------Command: python Wild_phenotype_classifier.py Input: - Wild individuals SNP matrix (232 samples) - Trained model from Step 5 - VC scores for wild individuals Output: Predicted phenotype frequencies per population Wild Population Predictions: - NORTH (n=76): 98% obligate diapause (univoltine) - HIGH (n=77): 46% obligate, 54% facultative - LOW (n=79): 41% obligate, 59% facultative (multivoltine potential) STEP 7: Functional Annotation and GO Enrichment------------------------------------------------Command: Rscript SNP_mapping_and_GO_enrichment_script.r Input: - Significant SNPs from GWAS and ML - Gene annotations - Gene lengths - Gene-to-GO mappings Output: - SNP-gene overlaps - Enriched GO terms - Biological pathways implicated in diapause Key Enriched Pathways (both GWAS and ML): - Chromosome organization (GO:0051276) - Endocytosis (GO:0006897) - Scavenger receptor activity (GO:0005044) ================================================================================REQUIRED SOFTWARE AND VERSIONS================================================================================ Bioinformatics Tools--------------------* ipyrad: v0.9.95 (RAD-seq assembly)* vcftools: v0.1.16 (VCF filtering)* plink: v1.90b6.21 (LD pruning)* Liftoff: v1.6.3 (gene annotation transfer)* Baypass: v2.41 (GWAS analysis)* STRUCTURE: v2.3 (population structure) R and R Packages----------------* R: v4.4.2* vcfR: v1.7.0.91* adegenet: v1.3.1* rrBLUP: v4.6.3* GenomicRanges (Bioconductor)* goseq: v1.60.0* pophelper: v2.3.1 Python and Python Packages---------------------------* Python: v3.8+* lightgbm: v4.5.0* optuna: v4.2.0* pandas: v2.1.4* numpy: v2.1.3* scikit-learn: v1.5.2* joblib: v1.4.2 ================================================================================DATA FILES REQUIRED================================================================================ Input Data Files (NOT INCLUDED)----------------These files should be obtained separately or generated from raw sequencing data: 1. Raw sequencing reads: Available at NCBI SRA (BioProject: PRJDB38037)2. Reference genome: Ips typographus genome assembly (Nadachowska-Brzyska et al., 2025)3. Gene annotations: Transferred from Powell et al. (2021) using Liftoff4. Phenotype data: Binary labels (0/1) for obligate/facultative diapause (determined experimentally)5. Gene-to-GO mapping: Gene IDs with associated GO terms6. Gene lengths: Gene IDs with exon-based lengths in base pairs Data Files Included in This Repository---------------------------------------* phenotype_pruned_filtered75missing_LD.vcf: Filtered SNPs for 296 phenotyped individuals* WILD.recode.vcf: Filtered SNPs for 232 wild individuals* pop_assign.txt: Population assignments for all samples ================================================================================KEY PARAMETERS AND THRESHOLDS================================================================================ VCF Filtering-------------* Missing data threshold: 25%* LD pruning: r² = 0.2, window = 50 SNPs, step = 10 SNPs* Final SNP count: 11,867 (after filtering from 134,410) Baypass GWAS------------* MCMC runs: 5 independent runs with different seeds* Burn-in: 2,500 iterations* Pilot runs: 20 (npilot)* Pilot length: 200 iterations* Association strength interpretation: - BF > 2: Weak association - BF > 6: Moderate association - BF > 10: Strong association LightGBM Machine Learning--------------------------* Cross-validation: 50-fold* Hyperparameter optimization: 2,000 Optuna trials* Binary classification threshold: 0.5* Missing data handling: Native LightGBM imputation* Feature encoding: 0 (homozygous ref), 2 (heterozygous), 3 (homozygous alt) GO Enrichment-------------* Background: All genes in RAD-seq dataset* Foreground: Genes with SNPs (BF > 6 for GWAS; non-zero importance for ML)* Test: Wallenius' noncentral hypergeometric distribution* Length bias correction: Included via goseq ================================================================================EXPECTED OUTPUTS AND INTERPRETATION================================================================================ GWAS Results------------* High BF SNPs: Strong candidates for diapause regulation* JHE locus: Top-ranked SNP suggests juvenile hormone pathway involvement* Moderate BF SNPs: Additional candidates for multi-locus architecture Machine Learning Results------------------------* High importance SNPs: Most informative for phenotype prediction* AUROC > 0.9: Excellent discriminative ability* Accuracy ~86%: Robust phenotype classification Wild Population Predictions----------------------------* High proportion of obligate diapause (>90%): Univoltine populations, lower outbreak risk* High proportion of facultative diapause (>50%): Multivoltine potential, higher outbreak risk* Intermediate proportions: Variable voltinism depending on environmental conditions ================================================================================CITATIONS================================================================================ Key Methods References----------------------* ipyrad: Eaton & Overcast (2020) Bioinformatics 36:2592-2594* vcftools: Danecek et al. (2011) Bioinformatics 27:2156-2158* plink: Purcell et al. (2007) Am J Hum Genet 81:559-575* Liftoff: Shumate & Salzberg (2021) Bioinformatics 37:1639-1643* Baypass: Gautier (2015) Genetics 201:1555-1579* LightGBM: Ke et al. (2017) Advances in Neural Information Processing Systems 30:3149-3157* goseq: Young et al. (2010) Genome Biology 11:R14 Reference Genome----------------* Powell et al. (2021) Communications Biology 4:1059* Nadachowska-Brzyska et al. (2025) in preparation ================================================================================HOW TO CITE THIS DATASET================================================================================ Recommended citation format: Palmieri, L., Dowle, E.J., Nadachowska-Brzyska, K., Schopf, A., Dobart, N., Stauffer, C., Schuler, H., Ragland, G.J., & Schebeck, M. (2025). Genomics and Machine Learning Uncover Diapause Mechanisms and Predict Voltinism in Ips typographus - Analysis Pipeline and Data [Data set]. Zenodo. https://doi.org/10.5281/zenodo.17543185 Last updated: November 2025