Abstract
Ancient DNA (aDNA) studies are frequently focused on the analysis of the mitochondrial DNA (mtDNA), which is much more abundant than the nuclear genome, hence can be better retrieved from ancient remains. However, postmortem DNA damage and contamination make the data analysis difficult because of DNA fragmentation and nucleotide alterations. In this regard, the assessment of the heteroplasmic fraction in ancient mtDNA has always been considered an unachievable goal due to the complexity in distinguishing true endogenous variants from artifacts. We implemented and applied a computational pipeline for mtDNA analysis to a dataset of 30 ancient human samples from an Iron Age necropolis in Polizzello (Sicily, Italy). The pipeline includes several modules from well-established tools for aDNA analysis and a recently released variant caller, which was specifically conceived for mtDNA, applied for the first time to aDNA data. Through a fine-tuned filtering on variant allele sequencing features, we were able to accurately reconstruct nearly complete (>88%) mtDNA genome for almost all the analyzed samples (27 out of 30), depending on the degree of preservation and the sequencing throughput, and to get a reliable set of variants allowing haplogroup prediction. Additionally, we provide guidelines to deal with possible artifact sources, including nuclear mitochondrial sequence (NumtS) contamination, an often-neglected issue in ancient mtDNA surveys. Potential heteroplasmy levels were also estimated, although most variants were likely homoplasmic, and validated by data simulations, proving that new sequencing technologies and software are sensitive enough to detect partially mutated sites in ancient genomes and discriminate true variants from artifacts. A thorough functional annotation of detected and filtered mtDNA variants was also performed for a comprehensive evaluation of these ancient samples.
Highlights
Genetic material recovered from ancient samples has particular characteristics due to degradations that occurred through time
In this kind of analysis, the realignment to a circular version of the mitochondrial DNA (mtDNA) by CircularMapper is not a mandatory step; we chose to perform read realignment to ensure the most accurate haplogroup assignment, since many phylogenetically informative positions can be found at the beginning and the end of the mtDNA reference sequence (Peltzer et al, 2016). schmutzi allowed to identify deamination patterns, which are generally used as markers for ancient DNA (aDNA), and detect recent mitochondrial contaminants by comparing each base position with the corresponding one in 197 known modern Eurasian mtDNA sequences (Renaud et al, 2015)
Since the analysis by schmutzi is a well-established practice in the aDNA field, we considered the 27 consensus sequences and the set of related variants detected in each sample as a reference to validate results obtained by our approach
Summary
Genetic material recovered from ancient samples has particular characteristics due to degradations that occurred through time. In many studies on ancient samples, mitochondrial DNA (mtDNA) has been preferred as a target because of its high number of copies in each cell and its higher availability with respect to nuclear DNA. It represents a useful marker for evolutionary and population genetics analysis extensively applied on ancient samples (Modi et al, 2017; Chylenski et al, 2019; Ehler et al, 2019; Modi et al, 2019, 2020a,b; Översti et al, 2019; Vai et al, 2019; Juras et al, 2020; Yang et al, 2020). Besides contamination by exogenous DNA samples, another source of artifacts generally affecting mtDNA analysis is represented by nuclear sequences of mitochondrial origin (NumtS), barely explored in aDNA (den Tex et al, 2010; Samaniego Castruita et al, 2015)
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