![]() They allow: (i) recovering short molecules, even <50 bp long, that can represent a considerable amount of the endogenous fraction in archeological samples ( Prüfer et al., 2010 Dabney et al., 2013) (ii) direct sequencing with no targeted amplification steps and (iii) generating high amounts of sequence data at low cost. Next-generation sequencing (NGS) technologies have revolutionized the field of aDNA. However, 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). It represents also a useful marker for evolutionary and population genetics analysis extensively applied on ancient samples ( Modi et al., 2017 Chyleński 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). 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 consequently its higher availability with respect to nuclear DNA. Fragmentation and decline of the amount of endogenous molecules are mainly due to depurination caused by hydrolysis while deamination of cytosines tends to occur at the 5′ ends, being translated in misincorporations C to T (C > T) at 5′ and G to A (G > A) at 3′ in the final sequence obtained from a double-stranded library ( Briggs et al., 2007). Degradation of the genetic material is due to factors such as temperature, pH, and processes as hydrolysis and oxidation that act on the biological sample through time. Endogenous ancient DNA (aDNA) molecules are often retrieved in low copy number, with the co-presence of possible exogenous contaminant DNA, and are characterized by high fragmentation and a typical pattern of damage at read termini. Genetic material recovered from ancient samples has particular characteristics due to degradations that occurred through time. A thorough functional annotation of detected and filtered mtDNA variants was also performed for a comprehensive evaluation of these ancient samples. 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. Additionally, we provide guidelines to deal with possible artifact sources, including nuclear mitochondrial sequence (NumtS) contamination, an often-neglected issue in ancient mtDNA surveys. 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. 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. 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). 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. However, postmortem DNA damage and contamination make the data analysis difficult because of DNA fragmentation and nucleotide alterations. 2Dipartimento di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche, Università degli Studi di Palermo, Palermo, ItalyĪncient 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.1Dipartimento di Biologia, Università degli Studi di Firenze, Florence, Italy. ![]() Maria Angela Diroma 1*† Alessandra Modi 1† Martina Lari 1 Luca Sineo 2 David Caramelli 1 Stefania Vai 1* ![]()
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |