Although most ancient DNA studies have focused on the last 50,000 years, paleogenomic approaches can now reach into the early Pleistocene, an epoch of repeated environmental changes that shaped present-day biodiversity. Emerging deep-time genomic transects, including from DNA preserved in sediments, will enable inference of adaptive evolution, discovery of unrecognized species, and exploration of how glaciations, volcanism, and paleomagnetic reversals shaped demography and community composition. In this Review, we explore the state-of-the-art in paleogenomics and discuss key challenges, including technical limitations, evolutionary divergence and associated biases, and the need for more precise dating of remains and sediments. We conclude that with improvements in laboratory and computational methods, the emerging field of deep-time paleogenomics will expand the range of questions addressable using ancient DNA.

We present a protocol to prepare extracted DNA for sequencing on the Illumina sequencing platform that has been optimized for ancient and degraded DNA. Our approach, the Santa Cruz Reaction or SCR, uses directional splinted ligation of Illumina's P5 and P7 adapters to convert natively single-stranded DNA and heat denatured double-stranded DNA into sequencing libraries in a single enzymatic reaction. To demonstrate its efficacy in converting degraded DNA molecules, we prepare 5 ancient DNA extracts into sequencing libraries using the SCR and 2 of the most commonly used approaches for preparing degraded DNA for sequencing: BEST, which targets and converts double-stranded DNA, and ssDNA2.0, which targets and converts single-stranded DNA. We then compare the efficiency with which each approach recovers unique molecules, or library complexity, given a standard amount of DNA input. We find that the SCR consistently outperforms the BEST protocol in recovering unique molecules and, despite its relative simplicity to perform and low cost per library, has similar performance to ssDNA2.0 across a wide range of DNA inputs. The SCR is a cost- and time-efficient approach that minimizes the loss of unique molecules and makes accessible a taxonomically, geographically, and a temporally broader sample of preserved remains for genomic analysis.

Several methods exist for detecting genetic relatedness or identity by comparing DNA information. These methods generally require genotype calls, either single-nucleotide polymorphisms or short tandem repeats, at the sites used for comparison. For some DNA samples, like those obtained from bone fragments or single rootless hairs, there is often not enough DNA present to generate genotype calls that are accurate and complete enough for these comparisons. Here, we describe IBDGem, a fast and robust computational procedure for detecting genomic regions of identity-by-descent by comparing low-coverage shotgun sequence data against genotype calls from a known query individual. At less than 1× genome coverage, IBDGem reliably detects segments of relatedness and can make high-confidence identity detections with as little as 0.01× genome coverage.

Hybridization capture approaches allow targeted high-throughput sequencing analysis at reduced costs compared to shotgun sequencing. Hybridization capture is particularly useful in analyses of genomic data from ancient, environmental, and forensic samples, where target content is low, DNA is fragmented and multiplex PCR or other targeted approaches often fail. Here, we describe a DNA bait synthesis approach for hybridization capture that we call Circular Nucleic acid Enrichment Reagent, or CNER (pronounced 'snare'). The CNER method uses rolling-circle amplification followed by restriction digestion to discretize microgram quantities of hybridization probes. We demonstrate the utility of the CNER method by generating probes for a panel of 23 771 known sites of single nucleotide polymorphism in the horse genome. Using these probes, we capture and sequence from a panel of ten ancient horse DNA libraries, comparing CNER capture efficiency to a commercially available approach. With about one million read pairs per sample, CNERs captured more targets (90.5% versus 66.5%) at greater mean depth than an alternative commercial approach.

The chance discovery of a 1.5-3.5 years old mummified girl presents a unique opportunity to further our understanding of health and disease among children in 19th Century San Francisco. This study focuses on carbon and nitrogen stable isotope signatures in serial samples of hair that cover the last 14 months of her life. Results suggest an initial omnivorous diet with little input from marine resources or C4 plants. Around six months before death δ15N starts a steady increase, with a noticeable acceleration just two months before she died. The magnitude of δ15N change, +1.5‰ in total, is consistent with severe undernourishment or starvation. Cemetery records from this time period in San Francisco indicate high rates of infant and child mortality, mainly due to bacterial-borne infectious diseases, about two orders of magnitude higher than today. Taken together, we hypothesize that the girl died after a prolonged battle with such an illness. Results highlight the tremendous impacts that modern sanitation and medicine have had since the 1800s on human health and lifespan in the United States.

Relict woolly mammoth (Mammuthus primigenius) populations survived on several small Beringian islands for thousands of years after mainland populations went extinct. Here we present multiproxy paleoenvironmental records to investigate the timing, causes, and consequences of mammoth disappearance from St. Paul Island, Alaska. Five independent indicators of extinction show that mammoths survived on St. Paul until 5,600 ± 100 y ago. Vegetation composition remained stable during the extinction window, and there is no evidence of human presence on the island before 1787 CE, suggesting that these factors were not extinction drivers. Instead, the extinction coincided with declining freshwater resources and drier climates between 7,850 and 5,600 y ago, as inferred from sedimentary magnetic susceptibility, oxygen isotopes, and diatom and cladoceran assemblages in a sediment core from a freshwater lake on the island, and stable nitrogen isotopes from mammoth remains. Contrary to other extinction models for the St. Paul mammoth population, this evidence indicates that this mammoth population died out because of the synergistic effects of shrinking island area and freshwater scarcity caused by rising sea levels and regional climate change. Degradation of water quality by intensified mammoth activity around the lake likely exacerbated the situation. The St. Paul mammoth demise is now one of the best-dated prehistoric extinctions, highlighting freshwater limitation as an overlooked extinction driver and underscoring the vulnerability of small island populations to environmental change, even in the absence of human influence.

The extinct 'New World stilt-legged', or NWSL, equids constitute a perplexing group of Pleistocene horses endemic to North America. Their slender distal limb bones resemble those of Asiatic asses, such as the Persian onager. Previous palaeogenetic studies, however, have suggested a closer relationship to caballine horses than to Asiatic asses. Here, we report complete mitochondrial and partial nuclear genomes from NWSL equids from across their geographic range. Although multiple NWSL equid species have been named, our palaeogenomic and morphometric analyses support the idea that there was only a single species of middle to late Pleistocene NWSL equid, and demonstrate that it falls outside of crown group Equus. We therefore propose a new genus, Haringtonhippus, for the sole species H. francisci. Our combined genomic and phenomic approach to resolving the systematics of extinct megafauna will allow for an improved understanding of the full extent of the terminal Pleistocene extinction event.

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa¹. However, the irreversible post-mortem degradation² of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)³. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I⁴, and suggested the presence of protein residues in fossils of the Cretaceous period⁵-although with limited phylogenetic use⁶. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch^7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)¹⁰. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates¹¹, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.