Nicholas W. VanKuren (1), Nathan P. Buerkle(1), Wei Lu, Erica L. Westerman, Alexandria K. Im, Darli Massardo, Laura Southcott, Stephanie E. Palmer, Marcus R. Kronforst

(1) Contributed equally to this manuscript. Contact Nick (nwvankuren@gmail.com;nvankuren@uchicago.edu) or Nathan (nathan.p.buerkle@gmail.com;nathan.buerkle@yale.edu) with questions about the supplied datasets or analysis. 

Access this dataset on Dryad

Abstract

This Dryad repository contains raw data, scripts, and notebooks that can recapitulate the key analyses performed in the associated publication and allow further exploration the results. This repository contains three main sections:

1. Data, scripts, and a notebook detailing the genome-wide association analyses for forewing color and male mate choice. Data includes:
   • Raw forewing color and courtship behavior data from Chamberlain et al. (2009).
   • Genome-wide variant calls using whole-genome re-sequencing data from 113 H. cydno alithea males.
   • Raw and intermediate analysis results for color and choice genome-wide association tests
   • Intermediate results for analyses of linkage disequilibrium and $F_{ST}$ statistics
   • The H. c. alithea (yellow) reference genome sequence and gene annotation generated and used in this paper
2. Data, scripts, and a notebook detailing the analysis of RNA-sequencing data for H. c. alithea and H. c. galanthus central brain, optic lobe, and retina at seven developmental stages. Data includes:
   • Intermediate gene expression data
   • Full results from differential expression analyses presented the paper
3. Data with the processed electrophysiology data for single photoreceptors labeled by species, sex, and individual
   • Spectral tuning curve response amplitudes and latencies
   • V-Log(I) response amplitudes and latencies
   • Presence or absence of long-wavelength inhibition
   • Effect of background, green LED on photoreceptor responses

Versions

• 2024-12-09 Update: 
  • Updated title to reflect final title.
  • Added a new directory, electrophysiology.zip , containing raw data from electrophysiology experiments and wing spectral reflectance data. Updated README to describe new files. 
  • Updated Methods section to describe electrophysiology data collection
• 2024-10-18 Update:
  • Updated title to reflect order of paper results.
  • Updated Methods section of this repository to add information of how data required for analyses described in the gwas directory were generated. 
  • Minor updates to initial rnaseq submission, including: minor text edits in notebook.Rmd for clarity; the addition of an updated file data/GenesInOtherPrefPeaks.50kb.2024-10-10.txt containing a list of genes found near genome-wide association (GWA) peaks on chromosomes 7, 9, and 11. 
  • Relocated top-level genome and genome annotation files into gwas/info/genome directory.
  • Added a new gwas directory containing data, scripts, and an R notebook required for running the GWA analyses presented in the associated publication, plus additional QC steps that were not presented in the publication but are important for understanding our final analyses. This README has been updated to reflect this addition and describes each of those files below. The gwas/notebook.Rmd R notebook contains additional information and will knit properly after installation of the required R packages. 

Description of the data and file structure

Description of data and files within the gwas directory:

The most important files in this directory are the notebook.Rmd and the gwas_notebook.Rproj files. These two files can together be used to re-run analyses presented in the paper, contain much of the detail and reasoning behind each analysis, and link all of the data and scripts described below into a single cohesive story. Please start there.

gwas.tar.gz

• gwas_notebook.Rproj: R project file for loading into R and RStudio. In conjunction with notebook.Rmd, can be used to re-run analyses presented in the associated paper and to run new analyses of interest to end user.
• notebook.Rmd: R markdown file containing detailed notes, code chunks, and additional data processing steps that can be used to recapitulate the paper results and allow exploration of the results further.
• expected_notebook.html: output from knitting notebook.Rmd before dryad submission. End users may simply browse this notebook or knit the project fresh.
• cluster_scripts/
  • PreprocessReads.sh: a shell script for SLURM that can be used to perform quality control and mapping of whole-genome re-sequencing data for the samples found in BioProject PRJNA802836
  • RunHaplotypeCaller.sh: a shell script SLURM that can be used to perform the first step of calling genome-wide variation in the alignment files generated by PreprocessReads.sh. This script depends on the GenomeAnalysisToolKit v4.2 and the HaplotypeCaller module.
  • RunGenotypeGvcfs.sh: a shell script for SLURM that can be used to perform the second step of calling genome-wide variation using the output from RunHaplotypeCaller.sh. This script depends on the GenomeAnalysisToolKit v4.2.
  • RunGemmaFwcGwa.sh: a shell script for SLURM that can be used to perform the genome-wide association for male forewing color (fwc) using the variant callset found in data/plink/heliconius_cydno_alithea_yellow.variants.20230712.pruned...
  • RunGemmaLmpGwa.sh: a shell script for SLURM that can be used to perform the genome-wide association for male lifetime mate preference (lmp) using the variant callset found in data/plink/heliconius_cydno_alithea_yellow.variants.20230712.pruned...
  • RunGmmatChoiceWald.R: an R script that will perform the genome-wide association for choice using GMMAT 1.4.2, the variant callset in data/plink/heliconius_cydno_alithea_yellow.variants.20230712.pruned... and the raw phenoytpe and choice data in data/phenotype_data/MalePhenotypeData.20190306.txt and data/phenotype_data/CourtsForR.20190506.EXPANDED.csv.
  • RunGmmatChoiceWald.sh: a shell script for SLURM that wraps RunGmmatChoiceWald.R. - RunGmmatFwcWald.R: an R script that will perform the genome-wide association for male forewing color (fwc) using GMMAT 1.4.2, the variant callset in data/plink/heliconius_cydno_alithea_yellow.variants.20230712.pruned... and the raw phenoytpe data in data/phenotype_data/MalePhenotypeData.20190306.txt.
  • RunGmmatFwcWald.sh: a shell script for SLURM that wraps RunGmmatFwcWald.R.
• current_cache/: an empty directory to contain cached results from knitting the R notebook ./notebook.Rmd
• data/
  • fst/
  • heliconius_cydno_alithea_yellow.variants.20230712.pruned.10k2k.windowed.weir.fst.gz: gzipped text file containing $F_{ST}$ calculations in 10 kb sliding windows (2 kb step) between yellow and white males. Calculated using vcftools 0.16 and the variant calls in data/plink/heliconius_cydno_alithea_yellow.variants.20230712.pruned...
  • gwas/
  • choice.grm_only.20240923.out.txt.gz: gzipped text file containing the genome-wide association results for male mate choice including only the genetic relatedness matrix (grm) as a random effect.
  • choice.grm_stock.20240925.out.txt.gz: gzipped text file containing the genome-wide association results for male mate choice including the genetic relatedness matrix (grm) and principal component 2 (pc2, or stock) as random effects.
  • forewing_color.cXX.txt: the genetic relatedness matrix calculated using GEMMA 0.98.5 and the variant callset in data/plink/heliconius_cydno_alithea_yellow.variants.20230712.pruned...
  • forewing_color.grm_only.assoc.txt.gz: gzipped text file containing the genome-wide association results for male forewing color including only the genetic relatedness matrix as a random effect.
  • forewing_color.grm_pc2.assoc.txt.gz: gzipped text file containing the genome-wide association results for male forewing color including the genetic relatedness matrix and principal component 2 (pc2, or stock) as random effects.
  • forewing_color.grm_pcs1-3.assoc.txt.gz: gzipped text file containing the genome-wide association results for male forewing color including the genetic relatedness matrix (grm) and principal components 1-3 as random effects.
  • klocus.court_num.20240926.out.txt.gz: gzipped text file containing the association results for male mate choice in the K locus (chr1:14.5Mb-16.5Mb) including the genetic relatedness matrix (grm) a random effect and court number as an additional fixed effect.
  • klocus.male_fwc.20240926.out.txt.gz: gzipped text file containing the association results for male mate choice in the K locus (chr1:14.5Mb-16.5Mb) including the genetic relatedness matrix (grm) a random effect and male forewing color as an additional fixed effect.
  • preference_all.grm_only.assoc.txt.gz: gzipped text file containing the genome-wide association results for male lifetime preference (estimated using all courts) and including only the genetic relatedness matrix as a random effect.
  • ld/
  • K_locus_LD.lt10kb.ld.gz: gzipped text file containing pairwise linkage disequilibrium (LD) estimates for all pairs of K locus variants less than 10 kb apart.
  • LdEstimates.100Mb.240905.txt: text file containing summaries of LD values for 600 million random pairs of variants from across the genome.
  • snp_ld_values: text file containing pairwise LD estimates for pairs of top forewing color and male mate choice variants identified using genome-wide association.
  • plink/
  • heliconius_cydno_alithea_yellow.variants.20230712.pruned.[bed|bim|fam|nosex]: plink bfile set containing filtered variant calls for all 113 males in the final dataset. This was the callset used for all genome-wide association analyses.
  • heliconius_cydno_alithea_yellow.variants.20230712.pruned.eigenv[ec|al]: text files containing principal components (PC) analysis results for the first 20 PCs. Calculated using PLINK 1.90 and the plink bfile set in this directory.
  • heliconius_cydno_alithea_yellow.variants.20230712.pruned.forewing_color.fam: plink fam file containing the male forewing color information.
  • susie/
  • SusieData.20241003.Rdata: R data file containing two objects used for the SuSiE analysis described in the publication and the notebook.
• info/
  • genome/
  • heliconius_cydno_alithea.yellow.fa.gz: bgzipped FASTA format file containing the yellow Heliconius cydno alithea genome sequence.
  • heliconius_cydno_alithea.yellow.agp: text file containing information linking heliconius_cydno_alithea.yellow.fa scaffolds to the Heliconius melpomene v2.5 genome assembly. Generated using RagTag and subsequent massaging with awk.
  • heliconius_cydno_alithea.yellow.genome: text file containing heliconius_cydno_alithea.yellow.fa scaffold names and lengths.
  • heliconius_cydno_alithea.yellow.decorated.modified.gff3.gz: gzipped generic feature format v3 (GFF3) file containing gene annotation information for the heliconius_cydno_alithea.yellow.fa genome sequence.
  • phenotype_data/
  • CourtsForR.20190506.csv: csv file containing raw courtship data from Chamberlain et al. (2009)
  • CourtsForR.20190506.EXPANDED.csv: csv file containing raw courtship data from Chamberlain et al. (2009) EXPANDED to include a single courtship event per row.
  • GenotypePhenotypeCourtshipDataForR.20190620.txt: tab-delimited file containing genotype and courtship data for males studied by Chamberlain et al. (2009)
  • MalePhenotypeData.20190306.txt: tab-delimited file containing male wing color pattern information from Chamberlain et al. (2009)
  • sequencing_qc/
  • Raw-Alithea-Data_multiqc_report[.html|_data.zip]: html file and associated data summarizing the quality of the raw whole-genome re-sequencing data using FastQC and MultiQC. Data can be downloaded from NCBI BioProject PRJNA802836.
  • Trimmed-Alithea-Data_multiqc_report[.html|_data.zip]: html file and associated data summarizing the quality of the processed whole-genome re-sequencing data using FastQC and MultiQC. Data can be downloaded from NCBI BioProject PRJNA802836 and processed using cluster_scripts/PreprocesReads.sh.
  • SequencingQcAndMappingForR.20190620.txt: text file containing summary data for re-sequenced samples. - plots/: empty directory for holding plots generated by knitting thenotebook.Rmd\ file in RStudio.
• scripts/
  • RunGmmatScore.R: R script to run the genome-wide assocation analysis for male mate choice using GMMAT and the score test.
  • functions_for_gwa.R: R script containing useful functions for loading data, plotting, and massaging data.

Description of data and files within the rnaseq directory:

The most important files in this directory are the notebook.Rmd and the rnaseq.Rproj files. These two files can together be used to re-run analyses presented in the paper, contain much of the detail and reasoning behind each analysis, and link all of the data and scripts described below into a single cohesive story. Please start there.

rnaseq.tar.gz

• rnaseq.Rproj: R project file for loading into RStudio
• notebook.Rmd: R markdown notebook containing detailed notes, pipelines, and code chunks for recapitulating the published results and to allow further independent exploration by the end user.
• data/
  • AllGeneFunctionalAnnotations.2023-04-04.csv.gz:
  • GenesInKlocus.2023-05-23.txt: A text file containing a list of genes within the K locus (Hcay201001o:14500000-16500000)
  • GenesInOtherPrefPeaks.50kb.2024-10-10.txt: A text file containing a list of genes within 50 kb of the top GWA peaks on chromosomes 7, 9, and 11.
  • heliconius_cydno_alithea_deseq_results.Rdata: R data file containing the results from all stage-specific differential expression analyses used in the publication. This is a list of tibbles, deseq_results, where each tibble contains the full results from a single stage-specific run of DESeq2. This can be used to analyze stage-specific DE results for any gene.
  • helicoinius_cydno_alithea.yellow.eggnog.csv.gz: A gzipped CSV file containing the results from running eggNOG's emapper utility on the full yellow Heliconius cydno alithea protein annotation set. This contains information of putative orthologs and functions for each gene.
  • heliconius_cydno_data.Rdata: An Rdata file containing four objects used to run the publication analyses.
  • tx2gene: a 2-column tibble containing transcript-to-gene mapping
  • sample_info: a tibble containing RNA-seq sample information
  • initial_dds: a DESeq2 data object containing unfiltered gene quantification data for each sample in sample_info. Values were loaded from raw salmon output for each sample using tximport package.
  • filtered_dds: a DESeq2 data object containing filtered gene quantification data for the final set of 260 samples included in the publication analyses. This is the initial_dds object filtered for genes with mean normalized expression values >50 and for non-outlier samples.
  • heliconius_cydno_DEGs.ym_v_wm.gFDR_0.01.Rdata: An Rdata file containing a list of vectors of DE genes, DEGs_list, found in each relevant DESeq2 and maSigPro comparison. This is deseq_results filtered to keep only genes with global FDR <= 0.01 for each comparison, plus genes found to be DE by maSigPro. See the notebook for details on how these vectors were generated.
  • heliconius_cydno_masigpro_results.Rdata: An Rdata file containing the results from maSigPro runs from each tissue. This file contains list objects for each tissue (central brain, optic lobe, and retina) with the raw maSigPro results. You can extract significant genes using maSigPro::get.siggenes().
  • heliconius_cydno_wgcna_objects.Rdata: An Rdata file containing the objects required for running and generated by WGCNA with the filtered_dds and sample_info information. This file contains:
  • datExpr: expression data, generated from DESeq2::counts(filtered_dds, norm = T)
  • datTraits: trait data, derived from sample_info
  • dynamicMEs: gene co-expression module eigenvectors (summaries of gene expression for all genes within that module), for dynamically pruned modules.
  • dynamicModuleColors: vector containing module colors for each gene included in datExpr. These are the module assignments given to each gene by running WGCNA.
  • dynamicModuleLabels: vector containing module labels (numbers) for each gene included in datExpr. These are the module assignments given to each gene by running WGCNA.
  • gene_tree: hierarchical clustering object (from hclust) describing the relationships of all genes in datExpr. You can plot using base::plot().
  • METree: hierarchical clustering object (from hclust) describing the relationships of all co-expressed gene modules, based on the eigenvectors contained in dynamicMEs. You can visualize using base::plot
• plots/publication_plots/rPCA outlier identification/: directory containing the rPCA outlier plots that we relied on for the publication's filtering and analysis steps. These may differ from the analogous plots generated by the notebook because of random seeding. Files are labeled 9-OutlierId.all_{stage}_{tissue}.outliers.png. Where stage and tissue are descrbed in the notebook.
• results/module_GOs/
  • go_enrichment/: directory containing topGO gene ontology (GO) enrichment results in CSV format for each co-expressed gene module identified in the publication analyses. Module colors match those in the dynamicModuleColors vector.
  • go_figure/: directory containing plots generated using GO-figure!, the results from WGCNA, and the gene annotations held in heliconius_cydno_alithea.yellow.eggnog.csv.gz. The results for each invocation are found in independent directories. The topgo_files directory contains the properly formatted input files, derived from the eggNOG annotations.
  • graphs/: empty directory to contain output from topGO runs produced during knitting.
• scripts/:
  • heliconius_RNAseq_functions.R: R script file containing custom functions that I used to produce plots, massage data, etc.
  • modified_masigpro_functions.R: R script file containing maSigPro functions that have been modified to produce more useable/aesthetically pleasing output. These supersede functions in the native maSigPro package when knitting this notebook.

Description of data and files within the electrophysiology directory:

electrophysiology.zip

• UV_photoreceptors.csv: Photoreceptor response data for all ultraviolet sensitive photoreceptors recorded.
• blue_photoreceptors.csv: Photoreceptor response data for all blue sensitive photoreceptors recorded.
• green_photoreceptors.csv: Photoreceptor response data for all green sensitive photoreceptors recorded.
• red_photoreceptors.csv: Photoreceptor response data for all red sensitive photoreceptors recorded.
• broadband_photoreceptors.csv: Photoreceptor response data for all broadband sensitive photoreceptors recorded.
• wing_reflectance.csv: Wing reflectance values and expected photoreceptor absorption based on an opsin tuning template.

Sharing/Access information

Raw whole genome sequencing data can be downloaded from the NCBI Short Read Archive through BioProject PRJNA802836.

Raw RNA-sequencing data can be downloaded from the NCBI SRA through BioProject PRJNA1019262.

Code/Software

R notebooks

All R work was performed in RStudio 2024.04.1+748 and R version 4.4.

R notebooks contained in the gwas and rnaseq directories contain all information needed to run and should install required packages upon first execution. The expected_notebook.html files contain the expected results from simply opening the notebooks in RStudio and knitting using knitr. Packages and version numbers are contained in those html files via sessionInfo().

All Rcode contained within the gwas and rnaseq directories should be self-contained and able to run on standard system setups. NWV executed R notebooks on a 2015 MacBook Pro with 16Gb RAM and 2.5GHz i7 processor running Monterey 12.7.5. W Lu executed on a Windows machine with 16Gb RAM, 2.4GHz i7 processor and running Windows 10.

R notebooks contain additional commands that we used to run standalone programs on a compute cluster or on the UNIX command line. These are contained within their own chunks within those notebooks. These chunks were used to produce many of the intermediate results files found in data, and the input and output to those commands is written relative to the respective directory.

SLURM scripts

It is likely that users will need to edit the SLURM directives in any of the cluster_scripts scripts for their particular setups, in particular the account and the module loading steps.

Additional programs

• GenomeAnalysisToolKit 4.2
• PLINK 1.90
• GEMMA 0.98.5
• samtools, bcftools, and htslib 1.20
• bowtie2 2.5.1
• trim_galore
• cutadapt 4.5
• FastQC 0.12.1
• MultiQC 1.21
• picard 2.8.1
• salmon 1.10.1
• GOFigure! 1.0.2