Identification of ancient remains through genomic sequencing

Abstract

Studies of ancient DNA have been hindered by the preciousness of remains, the small quantities of undamaged DNA accessible, and the limitations associated with conventional PCR amplification. In these studies, we developed and applied a genomewide adapter-mediated emulsion PCR amplification protocol for ancient mammalian samples estimated to be between 45,000 and 69,000 yr old. Using 454 Life Sciences (Roche) and Illumina sequencing (formerly Solexa sequencing) technologies, we examined over 100 megabases of DNA from amplified extracts, revealing unbiased sequence coverage with substantial amounts of nonredundant nuclear sequences from the sample sources and negligible levels of human contamination. We consistently recorded over 500-fold increases, such that nanogram quantities of starting material could be amplified to microgram quantities. Application of our protocol to a 50,000-yr-old uncharacterized bone sample that was unsuccessful in mitochondrial PCR provided sufficient nuclear sequences for comparison with extant mammals and subsequent phylogenetic classification of the remains. The combined use of emulsion PCR amplification and high-throughput sequencing allows for the generation of large quantities of DNA sequence data from ancient remains. Using such techniques, even small amounts of ancient remains with low levels of endogenous DNA preservation may yield substantial quantities of nuclear DNA, enabling novel applications of ancient DNA genomics to the investigation of extinct phyla.

Figure 1.

Emulsion PCR amplification of ancient DNA extracts. (A) PAGE gel electrophoresis of ancient DNA extracts pre- (−) and post-amplification (+), using GS20 or Illumina adapters. Each pre-amplification lane contains 1 ng of ancient DNA. Each post-amplification lane contains one-fifth the amplification product from 1 ng (* = 0.1 ng, Wolf, right-hand lane) of starting DNA; 100-bp DNA ladder is shown for size comparison. The lengths of GS20 adapters (88 bp) and Illumina adapters (90 bp) must be taken into consideration when calculating amplified fragment size range. (B) Emulsion PCR (em) and aqueous nonemulsion PCR (aq) amplification of ancient wolf DNA. Nonemulsion PCR consistently results in higher molecular weight products, presumably due to chimeric sequences formed during the amplification process.

Figure 2.

Sequence composition of amplified ancient DNA extracts. Extracts were sequenced by GS20 and aligned to mammalian genomes using BLAST. Sequences aligning to microbial sequences in NR or with no alignment are not shown.

Figure 3.

Sequence composition of amplified and unamplified ancient wolf DNA. Sequence composition was determined by BLAST analysis of 1486 capillary sequencing reads of clones from an unamplified ancient wolf DNA and 12,221 reads generated by GS20 sequencing of the amplified wolf DNA extract. The NR category includes any sequences that had a hit to the nonredundant nucleotide database, including those that aligned ambiguously to multiple database sequences.

Figure 4.

Distribution of ancient wolf Illumina sequences on the dog genome. The proportion of the dog genome contained on each chromosome (bars) is shown along with the proportion of ancient wolf sequences aligning to each dog chromosome (black line).

Figure 5.

Maximum-likelihood estimation of the phylogenetic relationship of ancient incisor DNA sequences to extant ruminants (including orthologous sequences from the cow reference genome bosTau2). (A) Phylogenetic reconstruction using 645-bp orthologous sequence from several ruminant species. Deer sequence was used to root the trees. (B) Phylogenetic reconstruction using a 1153-bp orthologous sequence from bovine species. Sheep sequence was used to root the trees; (*) seven out of nine and 12 out of 14 substitutions on the ancient incisor branch are C > T or A > G, consistent with ancient DNA damage. The total number of substitutions is shown for each branch, along with percent support for internal nodes.