MitoNumt: A Workflow for Recovering Mitochondrial Genomes From Long-Read Data and Detecting NUMT Contamination in...

Nuclear mitochondrial DNA segments (NUMTs) are genomic DNA fragments that originate from historical insertions of mitochondrial DNA sequences into the nuclear genome. During mitochondrial genome (mtgenome) assembly from short-read genome sequencing data, NUMT fragments may be misidentified and incorporated into mtgenome assemblies, leading to NUMT contamination. Although some assembly programs include steps to filter NUMTs, these approaches are not always effective. Here, we present MitoNumt, a multifunctional workflow. Using this pipeline, we recovered mtgenomes, complete NUMT (coexistence with the mtgenomes), and incomplete NUMT fragments from PacBio HiFi long-read sequencing data for five species of the genus Panthera and Puma concolor. We then used these sequences to check mtgenomes available in the NCBI database. NUMT contamination was detected in mtgenomes from all Panthera species examined, with 23 contaminated mtgenomes identified and contamination lengths ranging from 3 to 416 bp. In addition, phylogenetic trees reconstructed from NUMT fragments of at least 5000 bp consistently recovered a topology congruent with the currently accepted Panthera phylogeny. Overall, this workflow enables the identification of HiFi-supported mtgenomes and complete NUMTs, and the detection of NUMT contamination in assembled mtgenomes, thereby improving the reliability of mtgenome-based evolutionary and genetic studies.