▪ Abstract About 20 years ago, DNA sequences were separately described from the quagga (a type of zebra) and an ancient Egyptian individual. What made these DNA sequences exceptional was that they were derived from 140- and 2400-year-old specimens. However, ancient DNA research, defined broadly as the retrieval of DNA sequences from museum specimens, archaeological finds, fossil remains, and other unusual sources of DNA, only really became feasible with the advent of techniques for the enzymatic amplification of specific DNA sequences. Today, reports of analyses of specimens hundreds, thousands, and even millions of years old are almost commonplace. But can all these results be believed? In this paper, we critically assess the state of ancient DNA research. In particular, we discuss the precautions and criteria necessary to ascertain to the greatest extent possible that results represent authentic ancient DNA sequences. We also highlight some significant results and areas of promising future research.

The Great Hungarian Plain was a crossroads of cultural transformations that have shaped European prehistory. Here we analyse a 5,000-year transect of human genomes, sampled from petrous bones giving consistently excellent endogenous DNA yields, from 13 Hungarian Neolithic, Copper, Bronze and Iron Age burials including two to high (~22 × ) and seven to ~1 × coverage, to investigate the impact of these on Europe’s genetic landscape. These data suggest genomic shifts with the advent of the Neolithic, Bronze and Iron Ages, with interleaved periods of genome stability. The earliest Neolithic context genome shows a European hunter-gatherer genetic signature and a restricted ancestral population size, suggesting direct contact between cultures after the arrival of the first farmers into Europe. The latest, Iron Age, sample reveals an eastern genomic influence concordant with introduced Steppe burial rites. We observe transition towards lighter pigmentation and surprisingly, no Neolithic presence of lactase persistence.

We show that DNA molecules amplified by PCR from DNA extracted from animal bones and teeth that vary in age between 25 000 and over 50 000 years carry C-->T and G-->A substitutions. These substitutions can reach high proportions among the molecules amplified and are due to the occurrence of modified deoxycytidine residues in the template DNA. If the template DNA is treated with uracil N-glycosylase, these substitutions are dramatically reduced. They are thus likely to result from deamination of deoxycytidine residues. In addition, 'jumping PCR', i.e. the occurrence of template switching during PCR, may contribute to these substitutions. When DNA sequences are amplified from ancient DNA extracts where few template molecules initiate the PCR, precautions such as DNA sequence determination of multiple clones derived from more than one independent amplification are necessary in order to reduce the risk of determination of incorrect DNA sequences. When such precautionary measures are taken, errors induced by damage to the DNA template are unlikely to be more frequent than approximately 0.1% even under the unlikely scenario where each amplification starts from a single template molecule.