High-throughput sequencing technologies have revolutionized ancient DNA (aDNA) research by enabling the reconstruction of whole-genome sequences from traces of short and extremely degraded DNA fragments.
DNA preservation is highly variable across samples and environments, as well as within single archaeological remains. The current temporal range for whole-genome sequencing covers the past million years.
DNA extracts from ancient material are generally metagenomic assemblages that include the DNA from the host and its associated microorganisms, as well as from a range of environmental microorganisms that colonize the sample after its death.
Various molecular approaches have been developed to improve access to aDNA in samples and reduce the sequencing costs of paleogenomics. These include extraction methods tailored to ultrashort DNA fragments, target enrichment for library inserts annealing to a panel of nucleic acid probes, and library building procedures targeting each DNA strand individually or incorporating only the most damaged DNA fragments.
Analyses of aDNA are prone to contamination by modern DNA molecules, which generally show limited degradation and fragmentation. Therefore, ruling out contamination — for example, exploiting patterns of DNA degradation and monitoring the heterozygosity levels observed at haploid loci — represents the cornerstone of every aDNA study.
DNA degradation reactions taking place post-mortem introduce mutation and depth-of-coverage patterns in the sequence data that can be exploited to authenticate paleogenomes and reconstruct genome-wide nucleosome and methylation maps.
Research involving ancient DNA (aDNA) has experienced a true technological revolution in recent years through advances in the recovery of aDNA and, particularly, through applications of high-throughput sequencing. Formerly restricted to the analysis of only limited amounts of genetic information, aDNA studies have now progressed to whole-genome sequencing for an increasing number of ancient individuals and extinct species, as well as to epigenomic characterization. Such advances have enabled the sequencing of specimens of up to 1 million years old, which, owing to their extensive DNA damage and contamination, were previously not amenable to genetic analyses. In this Review, we discuss these varied technical challenges and solutions for sequencing ancient genomes and epigenomes.
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This work was supported by the Danish Council for Independent Research, Natural Sciences (FNU, 4002-00152B and 0602-02383B); the Danish National Research Foundation (DNFR94); the Lundbeck Foundation (R52-5062); a Marie Curie Career Integration Grant (FP7 CIG-293845); and the “Chaires d'Attractivité 2014” IDEX, University of Toulouse, France.
The authors declare no competing financial interests.
- Osseous materials
Calcified animal tissues, such as bones and teeth.
- Second-generation sequencing
High-throughput short-read DNA sequencing platforms that require library construction and thus modification of the DNA before sequencing. Most commonly represented by the Illumina, GS-FLX (454), ABI SOLiD and Ion Torrent series.
- Resonance structures
Dynamic, alternative forms of molecular groups, such as nucleotide bases, that result from electron delocalization within the molecule.
Exposure of ancient calcified materials to a short initial digestion aimed at removing substantial fractions of exogenous contaminants.
The initial generation of GS-FLX sequencing platforms based on pyrosequencing, before their acquisition and renaming by Roche.
- T/A ligation
A common DNA ligation technology that relies on complementary pairing of thymine and adenine overhangs at the 3′ ends of the adaptors and inserts to be ligated, respectively.
- Shotgun sequencing
The sequencing of fragmented DNA in the absence of any selection strategy.
- Primer extension capture
(PEC). An enrichment technology based on the ligation of short 5′-biotinylated oligonucleotides (including a 12-nucleotide-long spacer followed by a primer of 18–25 nucleotides that is designed to match a particular region of interest) to single-stranded target molecules. This is followed by a single round of polymerase-based extension so as to increase the length over which the molecules are hybridized.
- Tiled probes
Probes that overlap in their positioning on the target so as to ensure that every target position is covered by more than one different probe.
- Chimeric DNA libraries
Recombination between libraries containing different template molecules during library PCR amplification, resulting in hybrid (chimeric) sequences that do not represent true biological sequences.
- Double-indexed DNA libraries
DNA libraries in which short (for example, 8 bp long) unique nucleotide indexes are incorporated within both adaptors used during library construction. Indexes are bordered by known sequences that serve to prime index sequencing reactions and also enable library attachment to the surface of the sequencing flow cell.
- Mate pairs
Pairs of sequences derived from both ends of a DNA library.
- Edit distance
The number of sequence mismatch counts between reads and targets.
- Probabilistic aligners
Mapping algorithms that can accommodate non-uniform distributions of sequencing errors along reads, generally leading to improved alignments between reads and reference genomes.
- Thermal age
The predicted time that it would have taken an archaeological sample to produce the observed degree of DNA degradation were the sample exposed to a constant temperature of 10 °C since deposition. Thermal age has been proposed to adjust the chronological age of a sample to its thermal history and to help in predicting the likelihood of DNA surviving in archaeological remains.
The DNA sequences of haploid chromosomes.
- Derived alleles
Alleles that are evolutionarily derived in a lineage of interest and that are not represented in an ancestral population or species.
- Ancestral alleles
Alleles in the ancestral state before a mutation took place in a descending population, species or lineage.
- Nearly fixed
Fixed alleles are those that are derived and present in all individuals in a descendent population or species. Nearly fixed alleles therefore represent those that are present in nearly all individuals (thus close to fixation, for example, showing allelic frequencies of 99% in the population).
Allelic variants showing identical genetic sequences but different epigenetic marks, such as different methylation patterns.
- Ghost population
An unsampled population that exchanges migrants with other sampled populations and that can be identified based on admixture signatures left in descending populations.
- Introgressive block lengths
Population admixture introduces a mosaic of ancestry blocks along the genome, the lengths of which decrease with each subsequent generation owing to recombination. Introgressive block lengths can therefore be exploited to determine the date of admixture events.
- CTCF regions
Genomic regions targeted by CCCTC-binding factor (CTCF) and involved in regulating the three-dimensional structure of chromatin and transcription by mediating long-range interactions between genomic sequences.
Interbreeding of individuals from multiple population origins, resulting in the introduction of DNA from one population into the genomes of a second population.
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Orlando, L., Gilbert, M. & Willerslev, E. Reconstructing ancient genomes and epigenomes. Nat Rev Genet 16, 395–408 (2015). https://doi.org/10.1038/nrg3935
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