Artifacts in single-cell mitochondrial DNA mutation analyses misinform phylogenetic inference

Sequencing mitochondrial DNA (mtDNA) variants from single cells has resolved clonality and lineage in native human samples and clinical specimens. Prior work established that heteroplasmic mtDNA variants can be used to delineate clonality in hematopoiesis, but they have limited ability to reconstruct cellular phylogenies. However, a recent report by Weng et al. challenges the current paradigm by describing an unprecedented number of shared mtDNA variants between cells that reportedly resolve high-resolution phylogenetic trees. We re-examined the claims of Weng et al., and identified two major points of concern regarding this unprecedented connectedness. First, shared variants between cells are disproportionately detected in a single molecule per cell, and second, these variants are enriched 10-20-fold at the edges of mtDNA molecules, reminiscent of artifacts reported in other sequencing approaches. Further, our analyses show that pruning low support and likely artificial mtDNA variants removes nearly all of the reported phylogenetic structure. Thus, we strongly caution against using mtDNA variant calling workflows that rely on minimal evidence, including the computational pipeline introduced in Weng et al., as variants with high connectedness and low evidence are likely artifacts that lead to the construction of false phylogenies.