Separating endogenous ancient DNA from modern day contamination in a Siberian Neandertal

Abstract

One of the main impediments for obtaining DNA sequences from ancient human skeletons is the presence of contaminating modern human DNA molecules in many fossil samples and laboratory reagents. However, DNA fragments isolated from ancient specimens show a characteristic DNA damage pattern caused by miscoding lesions that differs from present day DNA sequences. Here, we develop a framework for evaluating the likelihood of a sequence originating from a model with postmortem degradation-summarized in a postmortem degradation score-which allows the identification of DNA fragments that are unlikely to originate from present day sources. We apply this approach to a contaminated Neandertal specimen from Okladnikov Cave in Siberia to isolate its endogenous DNA from modern human contaminants and show that the reconstructed mitochondrial genome sequence is more closely related to the variation of Western Neandertals than what was discernible from previous analyses. Our method opens up the potential for genomic analysis of contaminated fossil material.paleogenomics | human evolution R etrieval and sequencing of DNA from fossil material has been revolutionized by the rapid surge of high-throughput sequencing technology (1). One of the advantages of the new generation of sequencing technologies over previous PCR-based approaches is the routine sequencing of molecules across their entire length in contrast to specific priming sites. Although this approach has been extended to ancient material from a number of organisms, there is particular interest in genomic analyses of ancient human populations (2-9). However, despite extensive precautions against contamination during laboratory sample preparation, many fossil samples show evidence of contamination from not only the microenvironment of the fossil, but also present day humans (2,(10)(11)(12)(13)(14). Although the former type of contamination can introduce statistical noise in comparisons of ancient DNA with modern populations, the latter type of contamination (from present day humans) can impose major biases on population genetic and phylogenetic analyses (15,16).Ancient DNA sequences show several features indicative of postmortem degradation (PMD) (17). The main diagnostic feature documented so far is a pattern of Cytosine (C) →Thymine (T) substitutions (18-20) that increases toward the 5′ end of the sequence reads (21), which in most applications, results in a complementary Guanine (G) →Adenine (A) pattern in the 3′ end caused by enzymatic repair (5). This pattern has been attributed to Cytosine deamination at single-stranded ends of the molecules (21) and shows a clear tendency of increase over time in contrast to other potential diagnostic features, such as fragment length and preferential fragmentation at purines (22,23). However, although the observation of such a pattern suggests the presence of degraded DNA in a sequence dataset, it does not prove that modern contamination is absent or even at low frequency. For high-coverage data from extinct close relatives of moder...

Nuclear and mitochondrial DNA sequences from two Denisovan individuals

“…It is clear that although these methods yield different contamination estimates, they all suggest that the nuclear DNA contamination in both libraries is substantial, particularly in Denisova 4, where it is likely to exceed 50%. To reduce the influence of DNA contamination (18,19), we therefore restrict the analyses of nuclear DNA to fragments that carry thymine residues at the first and/or last two positions at sites where the human reference sequence carries cytosine residues (but remove these C/T sites themselves in the analyses). Using these criteria, a total of 1.0 Mb of nuclear DNA sequences for Denisova 4 and 24.1 Mb for Denisova 8 (Table 1 and SI Appendix, Table S3) can be analyzed.…”

Almost 20 years of Neanderthal palaeogenetics: adaptation, admixture, diversity, demography and extinction

“…In subsequent studies, these patterns allowed the identification of authentic Neanderthal sequences and opened up the possibility of analysing Neanderthal samples that were previously discarded for genetic studies due to their high level of present-day human contamination [18].…”

“…At present, contamination is efficiently estimated, but only two in silico approaches have been developed to putatively separate endogenous from contaminant material [18,70]. However, neither of them precludes the sequencing of contaminant material, which might not be suitable if a high number of poorly handled and preserved samples have to be screened.…”

Selective enrichment of damaged DNA molecules for ancient genome sequencing

“…These substitutions can thus be taken as evidence for the presence of authentic ancient human sequences. Deamination-induced substitutions have also been exploited for separating ancient sequences from presentday human contamination in silico (Skoglund et al 2012(Skoglund et al , 2014aMeyer et al 2014;Raghavan et al 2014). Although effective in principle, this approach is costly, because a large proportion of sequence data is excluded from downstream analysis.…”