Methods for Large-Scale Intraspecific Analyses of Single Nucleotide Polymorphisms in the Mitochondrial Genomes of Aspergillus fumigatus.

Mitochondrial genomes (mitogenomes) are widely used in population genetics and evolutionary studies due to their high copy number per cell, conserved gene content, and informative sequence variation. In fungi, mitogenomes are particularly dynamic in their genome size and inheritance, making them valuable yet underutilized tools in genomic analyses. Here, we present a comprehensive, scalable protocol for large-scale mitochondrial population genomics using publicly available whole genome sequencing data from Aspergillus fumigatus, a ubiquitous mold and critical-priority human fungal pathogen. Leveraging the large collection of sequencing data from the NCBI Sequence Read Archive, this protocol enables high-throughput retrieval, processing, and analyses of mitogenome-wide single-nucleotide polymorphisms (mtSNPs) across thousands of isolates. Our workflow includes detailed steps for data acquisition, sequence read preprocessing, reference-based alignment, variant calling, and quality filtering. The data were then analyzed using workflows for phylogenetic inference, molecular and geographic population structure assessment, and recombination detection. We also introduce strategies for comparing mitochondrial to nuclear genome coverage ratios as a proxy for cellular genomic composition. The pipeline is optimized for high-performance computing environments and incorporates robust tools, such as GATK, BWA, BCFtools, and R packages, including poppr and adegenet. Though demonstrated with A. fumigatus, this protocol is adaptable to other eukaryotic species and provides a versatile framework for mitochondrial population genomic research across broad scales.