Iteratively mapping ancient<scp>DNA</scp>to reconstruct highly divergent mitochondrial genomes: An evaluation of software, parameters and bait reference

The ability to obtain ancient DNA (aDNA) from extinct species has greatly improved our study their evolutionary histories. Most aDNA studies focus on mitochondrial for both population-level and species-level insights, due its relatively high abundance increased obtainability. Although several approaches are available assemble mitogenomes, levels of damage non-target contaminating make many unsuitable aDNA. Further complicating matters most is the lack a sufficiently closely related extant relative act as reference. Iterative mapping can mitogenomes without using close reference sequence, been applied various species. Despite widespread use, accuracy reconstructed assemblies yet be comprehensively assessed. Here, we investigated influence (i) software (Burrows–Wheeler Alignment [BWA], MITObim NOVOplasty), (ii) parameters (iii) bait phylogenetic distance assembly two simulated datasets: spotted hyena mammalian references; southern cassowary avian references, empirical cave hyena; sloth Specifically, assessed results through three parameters: pairwise conspecific mitogenome, number incorrectly inserted base pairs total length assembly. We found large discrepancies in different software, references. Results also varied depending dataset, thereby making broad, generalised conclusions difficult. However, overall accurate were obtained with either BWA minimum quality 20 relaxed mismatch (-n 0.001 -o 2), or value 3 5. show could further by combining multiple Our provides information how divergent best By obtaining reconstruction possible, one more confident reliability downstream analyses, inferences made them.