Date Published: April 19, 2017
Publisher: Public Library of Science
Author(s): Endre Neparáczki, Klaudia Kocsy, Gábor Endre Tóth, Zoltán Maróti, Tibor Kalmár, Péter Bihari, István Nagy, György Pálfi, Erika Molnár, István Raskó, Tibor Török, David Caramelli.
As part of the effort to create a high resolution representative sequence database of the medieval Hungarian conquerors we have resequenced the entire mtDNA genome of 24 published ancient samples with Next Generation Sequencing, whose haplotypes had been previously determined with traditional PCR based methods. We show that PCR based methods are prone to erroneous haplotype or haplogroup determination due to ambiguous sequence reads, and many of the resequenced samples had been classified inaccurately. The SNaPshot method applied with published ancient DNA authenticity criteria is the most straightforward and cheapest PCR based approach for testing a large number of coding region SNP-s, which greatly facilitates correct haplogroup determination.
Comparing ancient DNA (aDNA) sequences extracted from well dated archaeological remains from different periods and locations provide crucial information about past human population history (reviewed in ). Phylogeographic inferences are drawn from phylogenetic and population genetic analyses of sequence variations, the quality of which can be biased by data quantity and quality. Nowadays Next Generation Sequencing technology (NGS) provides a growing number of high quality aDNA sequence data, but until recently the majority of aDNA studies have been restricted to short fragments from the hypervariable region-1 (HVR-I) of the mitochondrial DNA (mtDNA) genome, using PCR based methods. PCR based methods are very sensitive for contamination, as low amounts of exogenous DNA can easily dominate PCR products resulting in the recovery of irrelevant sequences [2–5]. As a result, in spite of the applied authenticity criteria , many of the published databases may contain unreliable sequences, which distort statistical analyses. This problem is especially relevant for many of the ancient populations, from which only PCR based HVR data are available.
As multicopy mtDNA is best preserved in archaeological remains than low copy nuclear DNA, most ancient sequences are derived from mitochondria . Within mtDNA, the most polymorphic HVR control region contains outstanding phylogenetic information, therefore HVR sequencing has been the primary method of choice for mtDNA hapolotyping. However HVR polymorphisms have a limited reliability for haplogroup determination, therefore in addition several informative coding region SNP-s (CR-SNP) were selected to unambiguously define haplogroups . At the beginning individual CR-SNP-s were determined with RFLP  or direct sequencing of PCR clones, but soon multiplex PCR combined with the SNaPshot technique  offered a more straightforward solution for identifying multiple SNP-s simultaneously. Latter method was soon adapted in the ancient DNA field  .