Abstract
BackgroundDespite the successful retrieval of genomes from past remains, the prospects for human palaeogenomics remain unclear because of the difficulty of distinguishing contaminant from endogenous DNA sequences. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks.Methodology/Principals FindingsTo investigate whether this pattern is present in ancient remains from a temperate environment, we have 454-FLX pyrosequenced different samples dated between 5,500 and 49,000 years ago: a bone from an extinct goat (Myotragus balearicus) that was treated with a depurinating agent (bleach), an Iberian lynx bone not subjected to any treatment, a human Neolithic sample from Barcelona (Spain), and a Neandertal sample from the El Sidrón site (Asturias, Spain). The efficiency of retrieval of endogenous sequences is below 1% in all cases. We have used the non-human samples to identify human sequences (0.35 and 1.4%, respectively), that we positively know are contaminants.ConclusionsWe observed that bleach treatment appears to create a depurination-associated fragmentation pattern in resulting contaminant sequences that is indistinguishable from previously described endogenous sequences. Furthermore, the nucleotide composition pattern observed in 5′ and 3′ ends of contaminant sequences is much more complex than the flat pattern previously described in some Neandertal contaminants. Although much research on samples with known contaminant histories is needed, our results suggest that endogenous and contaminant sequences cannot be distinguished by the fragmentation pattern alone.
Highlights
Ancient DNA analysis on extinct human populations can potentially provide information on past human migrations and evolutionary processes. aDNA extracts are composed, in variable ratios, of endogenous DNA and exogenous DNA
We observed that bleach treatment appears to create a depurination-associated fragmentation pattern in resulting contaminant sequences that is indistinguishable from previously described endogenous sequences
The adoption of several laboratory practices were advocated as a means to ensure the authenticity of aDNA results [1]. These included, for instance, physical separation of the ancient and modern DNA laboratory, frequent bleaching and UV irradiation on the working surfaces, the use of sterile laboratory wear, cloning of PCR products, and the independent replication of the results in a second laboratory. All these authentication criteria have proven to be ineffective while working on ancient human specimens, because modern human contaminant DNA can be mistaken for endogenous DNA [2]
Summary
Ancient DNA (aDNA) analysis on extinct human populations can potentially provide information on past human migrations and evolutionary processes. aDNA extracts are composed, in variable ratios, of endogenous DNA (either damaged or undamaged) and exogenous DNA. The adoption of several laboratory practices were advocated as a means to ensure the authenticity of aDNA results [1] These included, for instance, physical separation of the ancient and modern DNA laboratory, frequent bleaching and UV irradiation on the working surfaces, the use of sterile laboratory wear (including gloves and face masks), cloning of PCR products, and the independent replication of the results in a second laboratory. All these authentication criteria have proven to be ineffective while working on ancient human specimens, because modern human contaminant DNA can be mistaken for endogenous DNA [2]. Previous sequence data generated on high-throughput sequencing platforms indicate that fragmentation of ancient DNA sequences is a characteristic trait primarily arising due to depurination processes that create abasic sites leading to DNA breaks
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