We present a DNA library preparation method that has allowed us to reconstruct a high coverage (30X) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of “missing evolution” in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans.

Although an inverse relationship is expected in ancient DNA samples between the number of surviving DNA fragments and their length, ancient DNA sequencing libraries are strikingly deficient in molecules shorter than 40 bp. We find that a loss of short molecules can occur during DNA extraction and present an improved silica-based extraction protocol that enables their efficient retrieval. In combination with single-stranded DNA library preparation, this method enabled us to reconstruct the mitochondrial genome sequence from a Middle Pleistocene cave bear (Ursus deningeri) bone excavated at Sima de los Huesos in the Sierra de Atapuerca, Spain. Phylogenetic reconstructions indicate that the U. deningeri sequence forms an early diverging sister lineage to all Western European Late Pleistocene cave bears. Our results prove that authentic ancient DNA can be preserved for hundreds of thousand years outside of permafrost. Moreover, the techniques presented enable the retrieval of phylogenetically informative sequences from samples in which virtually all DNA is diminished to fragments shorter than 50 bp.T race amounts of DNA can occasionally survive the decomposition of organic matter for long periods of time after the death of an organism. However, the retrieval of these ancient DNA molecules is severely impeded by their small size. DNA fragmentation is at least partly driven by depurination (1, 2), a continually occurring process. It is thus predicted that the degree of DNA fragmentation increases with sample age. This correlation has, in fact, been established in a recent study that analyzed samples of different ages from the same archeological sites (3), but the correlation vanishes in comparisons across different sites (4). The important role of environmental conditions, especially temperature, in DNA preservation is well recognized and reflected-for example, in the concept of thermal age (5). Unsurprisingly, permafrost environments have yielded the oldest credible records of DNA survival, including short stretches of plant and invertebrate DNA with an estimated age of up to 800,000 y that were amplified by PCR from Artic ice cores (6, 7) and the genome sequence of a 700,000-y-old horse published recently (8). More temperate environments have yielded many DNA sequences from the Holocene and the Late Pleistocene, some as old as ∼100,000 (9) or ∼120,000 y (10), but only a single study has convincingly raised the possibility of DNA survival extending far into the Middle Pleistocene outside of permafrost (11). In this study, short PCR products of ∼50 bp were retrieved from several bone samples of Middle Pleistocene cave bears from European caves, the oldest coming from the site of Sima de los Huesos (Atapuerca, Spain) and estimated to be >300,000 y old.It is important to note that direct PCR amplification provides limited power to reconstruct sequences from short DNA fragments, because only fragments that are long enough to allow for the hybridization of two PCR primers around a stretch of informative sequence are amena...

Under favorable conditions DNA can survive for thousands of years in the remains of dead organisms. The DNA extracted from such remains is invariably degraded to a small average size by processes that at least partly involve depurination. It also contains large amounts of deaminated cytosine residues that are accumulated toward the ends of the molecules, as well as several other lesions that are less well characterized.

High-throughput sequencing technologies frequently necessitate the use of PCR for sequencing library amplification. PCR is a sometimes enigmatic process and is known to introduce biases. Here we perform a simple amplification-sequencing assay using 10 commercially available polymerase-buffer systems to amplify libraries prepared from both modern and ancient DNA. We compare the performance of the polymerases with respect to a previously uncharacterized template length bias, as well as GC-content bias, and find that simply avoiding certain polymerase can dramatically decrease the occurrence of both. For amplification of ancient DNA, we found that some commonly used polymerases strongly bias against amplification of endogenous DNA in favor of GC-rich microbial contamination, in our case reducing the fraction of endogenous sequences to almost half.

We present the DNA sequence of 17,367 protein-coding genes in two Neandertals from Spain and Croatia and analyze them together with the genome sequence recently determined from a Neandertal from southern Siberia. Comparisons with present-day humans from Africa, Europe, and Asia reveal that genetic diversity among Neandertals was remarkably low, and that they carried a higher proportion of amino acid-changing (nonsynonymous) alleles inferred to alter protein structure or function than present-day humans. Thus, Neandertals across Eurasia had a smaller long-term effective population than present-day humans. We also identify amino acid substitutions in Neandertals and present-day humans that may underlie phenotypic differences between the two groups. We find that genes involved in skeletal morphology have changed more in the lineage leading to Neandertals than in the ancestral lineage common to archaic and modern humans, whereas genes involved in behavior and pigmentation have changed more on the modern human lineage.ancient DNA | exome capture | site frequency spectra | paleogenetics A nalyses of the coding regions of multiple present-day human individuals have uncovered many amino acid-changing SNPs segregating at low frequency in present-day human populations (1-7). It also has been shown that Europeans carry a larger proportion than Africans of amino acid-changing SNPs inferred to alter the structure or function of proteins and thus to be potentially deleterious (4), a fact attributed to the bottleneck and subsequent expansion of human populations during and after the migration out of Africa (4). In contrast, little is known about the coding variation in Neandertals, an extinct hominin group closely related to presentday humans. The main reasons for this are the rarity of Neandertal remains and the fact that >99% of the DNA extracted from typical Neandertal bones is derived from microbes (8, 9), making shotgun sequencing of the endogenous DNA impractical.Here, we use a hybridization approach to enrich and sequence the protein-coding fractions of the genomes of two Neandertals from Spain and Croatia. We analyze them together with the genome sequence recently determined from a Neandertal from southern Siberia (10) and show that the genetic diversity, pattern of coding variation, and genes that may underlie phenotypic changes in Neandertals are remarkably different from those in present-day humans. Results and DiscussionWe used densely tiled oligonucleotide probes (9) and a recently described protocol (11) to capture the protein-coding exons from 17,367 genes in a ∼49,000-y-old (12) (uncalibrated radiocarbon date) Neandertal from Spain (SD1253, El Sidrón Cave) and a ∼44,000-y-old (uncalibrated radiocarbon date) Neandertal from Croatia (Vi33.15, Vindija Cave). The DNA libraries from these two Neandertals contain 0.2% and 0.5% endogenous DNA, respectively, and the capture approach enriched the endogenous DNA 325-fold and 153-fold, resulting in an average coverage of 12.5-fold and 42.0-fold for the El Sidrón and Vindi...

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Mycobacterium tuberculosis ( M. tb ), an obligate human pathogen and the etiological agent of tuberculosis (TB), remains a major threat to global public health. Comparative genomics has been invaluable for monitoring the emergence and spread of TB and for gaining insight into adaptation of M. tb . Most genomic studies of M. tb are based on single bacterial isolates that have been cultured for several weeks in vitro . However, in its natural human host, M. tb comprises complex, in some cases massive bacterial populations that diversify over the course of infection and cannot be wholly represented by a single genome. Recently, enrichment via hybridization capture has been used as a rapid diagnostic tool for TB, circumventing culturing protocols and enabling the recovery of M. tb genomes directly from sputum. This method has further applicability to the study of M. tb adaptation, as it enables a higher resolution and more direct analysis of M. tb genetic diversity within hosts with TB. Here we analyzed genomic material from M. tb and Mycobacterium bovis populations captured directly from sputum and from cultured samples using metagenomic and Pool-Seq approaches. We identified effects of sampling, patient, and sample type on bacterial genetic diversity. Bacterial genetic diversity was more variable and on average higher in sputum than in culture samples, suggesting that manipulation in the laboratory reshapes the bacterial population. Using outlier analyses, we identified candidate bacterial genetic loci mediating adaptation to these distinct environments. The study of M. tb in its natural human host is a powerful tool for illuminating host pathogen interactions and understanding the bacterial genetic underpinnings of virulence.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset neuromuscular condition caused by an abnormal polyglutamine (polyQ) tract expansion in androgen receptor (AR) protein. SBMA is a disease with high unmet clinical need. Recent studies have shown that mutant AR-altered transcriptional activity is key to disease pathogenesis. Restoring the transcriptional dysregulation without affecting other AR critical functions holds great promise for the treatment of SBMA and other AR-related conditions; however, how this targeted approach can be achieved and translated into a clinical application remains to be understood. Here, we characterized the role of AR isoform 2, a naturally occurring variant encoding a truncated AR lacking the polyQ-harboring domain, as a regulatory switch of AR genomic functions in androgen-responsive tissues. Delivery of this isoform using a recombinant adeno-associated virus vector type 9 resulted in amelioration of the disease phenotype in SBMA mice by restoring polyQ AR-dysregulated transcriptional activity.