Reconstructing the genetic history of late Neanderthals.

Although it has previously been shown that Neanderthals contributed DNA to modern humans, not much is known about the genetic diversity of Neanderthals or the relationship between late Neanderthal populations at the time at which their last interactions with early modern humans occurred and before they eventually disappeared. Our ability to retrieve DNA from a larger number of Neanderthal individuals has been limited by poor preservation of endogenous DNA and contamination of Neanderthal skeletal remains by large amounts of microbial and present-day human DNA. Here we use hypochlorite treatment of as little as 9 mg of bone or tooth powder to generate between 1- and 2.7-fold genomic coverage of five Neanderthals who lived around 39,000 to 47,000 years ago (that is, late Neanderthals), thereby doubling the number of Neanderthals for which genome sequences are available. Genetic similarity among late Neanderthals is well predicted by their geographical location, and comparison to the genome of an older Neanderthal from the Caucasus indicates that a population turnover is likely to have occurred, either in the Caucasus or throughout Europe, towards the end of Neanderthal history. We find that the bulk of Neanderthal gene flow into early modern humans originated from one or more source populations that diverged from the Neanderthals that were studied here at least 70,000 years ago, but after they split from a previously sequenced Neanderthal from Siberia around 150,000 years ago. Although four of the Neanderthals studied here post-date the putative arrival of early modern humans into Europe, we do not detect any recent gene flow from early modern humans in their ancestry.

Pretreatment: Removing DNA Contamination from Ancient Bones and Teeth Using Sodium Hypochlorite and Phosphate.

DNA isolated from ancient bones and teeth comprises a mixture of microbial contamination and DNA from the organism under study. In addition, analyses of ancient human remains are often complicated by contamination with present-day human DNA, which can be introduced during excavation and subsequent handling of the specimens. In most cases, the relative abundance of contaminant DNA is much greater than that of the target organism. Here we present two techniques for reducing the proportion of contaminant DNA in bones and teeth. The first and most efficient technique uses a sodium hypochlorite (bleach) pretreatment to destroy contaminant DNA that may be bound or otherwise attached to the surface of bone/tooth powder. The second, less destructive pretreatment uses a phosphate buffer to release surface-bound DNA.

A combined method for DNA analysis and radiocarbon dating from a single sample.

Current protocols for ancient DNA and radiocarbon analysis of ancient bones and teeth call for multiple destructive samplings of a given specimen, thereby increasing the extent of undesirable damage to precious archaeological material. Here we present a method that makes it possible to obtain both ancient DNA sequences and radiocarbon dates from the same sample material. This is achieved by releasing DNA from the bone matrix through incubation with either EDTA or phosphate buffer prior to complete demineralization and collagen extraction utilizing the acid-base-acid-gelatinization and ultrafiltration procedure established in most radiocarbon dating laboratories. Using a set of 12 bones of different ages and preservation conditions we demonstrate that on average 89% of the DNA can be released from sample powder with minimal, or 38% without any, detectable collagen loss. We also detect no skews in radiocarbon dates compared to untreated samples. Given the different material demands for radiocarbon dating (500 mg of bone/dentine) and DNA analysis (10-100 mg), combined DNA and collagen extraction not only streamlines the sampling process but also drastically increases the amount of DNA that can be recovered from limited sample material.

Reducing microbial and human contamination in DNA extractions from ancient bones and teeth.

Although great progress has been made in improving methods for generating DNA sequences from ancient biological samples, many, if not most, samples are still not amenable for analyses due to overwhelming contamination with microbial or modern human DNA. Here we explore different DNA decontamination procedures for ancient bones and teeth for use prior to DNA library preparation and high-throughput sequencing. Two procedures showed promising results: (i) the release of surface-bound DNA by phosphate buffer and (ii) the removal of DNA contamination by sodium hypochlorite treatment. Exposure to phosphate removes on average 64% of the microbial DNA from bone powder but only 37% of the endogenous DNA (from the organism under study), increasing the percentage of informative sequences by a factor of two on average. An average 4.6-fold increase, in one case reaching 24-fold, is achieved by sodium hypochlorite treatment, albeit at the expense of destroying 63% of the endogenous DNA preserved in the bone. While both pretreatment methods described here greatly reduce the cost of genome sequencing from ancient material due to efficient depletion of microbial DNA, we find that the removal of human DNA contamination remains a challenging problem.