Sequencing of target-enriched libraries is an efficient and cost-effective method for obtaining DNA sequence data from hundreds of nuclear loci for phylogeny reconstruction. Much of the cost of developing targeted sequencing approaches is associated with the generation of preliminary data needed for the identification of orthologous loci for probe design. In plants, identifying orthologous loci has proven difficult due to a large number of whole-genome duplication events, especially in the angiosperms (flowering plants). We used multiple sequence alignments from over 600 angiosperms for 353 putatively single-copy protein-coding genes identified by the One Thousand Plant Transcriptomes Initiative to design a set of targeted sequencing probes for phylogenetic studies of any angiosperm group. To maximize the phylogenetic potential of the probes, while minimizing the cost of production, we introduce a k-medoids clustering approach to identify the minimum number of sequences necessary to represent each coding sequence in the final probe set. Using this method, 5–15 representative sequences were selected per orthologous locus, representing the sequence diversity of angiosperms more efficiently than if probes were designed using available sequenced genomes alone. To test our approximately 80,000 probes, we hybridized libraries from 42 species spanning all higher-order groups of angiosperms, with a focus on taxa not present in the sequence alignments used to design the probes. Out of a possible 353 coding sequences, we recovered an average of 283 per species and at least 100 in all species. Differences among taxa in sequence recovery could not be explained by relatedness to the representative taxa selected for probe design, suggesting that there is no phylogenetic bias in the probe set. Our probe set, which targeted 260 kbp of coding sequence, achieved a median recovery of 137 kbp per taxon in coding regions, a maximum recovery of 250 kbp, and an additional median of 212 kbp per taxon in flanking non-coding regions across all species. These results suggest that the Angiosperms353 probe set described here is effective for any group of flowering plants and would be useful for phylogenetic studies from the species level to higher-order groups, including the entire angiosperm clade itself.

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SummarySmallpox holds a unique position in the history of medicine. It was the first disease for which a vaccine was developed and remains the only human disease eradicated by vaccination. Although there have been claims of smallpox in Egypt, India, and China dating back millennia [1, 2, 3, 4], the timescale of emergence of the causative agent, variola virus (VARV), and how it evolved in the context of increasingly widespread immunization, have proven controversial [4, 5, 6, 7, 8, 9]. In particular, some molecular-clock-based studies have suggested that key events in VARV evolution only occurred during the last two centuries [4, 5, 6] and hence in apparent conflict with anecdotal historical reports, although it is difficult to distinguish smallpox from other pustular rashes by description alone. To address these issues, we captured, sequenced, and reconstructed a draft genome of an ancient strain of VARV, sampled from a Lithuanian child mummy dating between 1643 and 1665 and close to the time of several documented European epidemics [1, 2, 10]. When compared to vaccinia virus, this archival strain contained the same pattern of gene degradation as 20th century VARVs, indicating that such loss of gene function had occurred before ca. 1650. Strikingly, the mummy sequence fell basal to all currently sequenced strains of VARV on phylogenetic trees. Molecular-clock analyses revealed a strong clock-like structure and that the timescale of smallpox evolution is more recent than often supposed, with the diversification of major viral lineages only occurring within the 18th and 19th centuries, concomitant with the development of modern vaccination.

Dogs were present in the Americas before the arrival of European colonists, but the origin and fate of these precontact dogs are largely unknown. We sequenced 71 mitochondrial and 7 nuclear genomes from ancient North American and Siberian dogs from time frames spanning ~9000 years. Our analysis indicates that American dogs were not derived from North American wolves. Instead, American dogs form a monophyletic lineage that likely originated in Siberia and dispersed into the Americas alongside people. After the arrival of Europeans, native American dogs almost completely disappeared, leaving a minimal genetic legacy in modern dog populations. The closest detectable extant lineage to precontact American dogs is the canine transmissible venereal tumor, a contagious cancer clone derived from an individual dog that lived up to 8000 years ago.

Molecular ecologists seek to genotype hundreds to thousands of loci from hundreds to thousands of individuals at minimal cost per sample. Current methods, such as restriction-site-associated DNA sequencing (RADseq) and sequence capture, are constrained by costs associated with inefficient use of sequencing data and sample preparation. Here, we introduce RADcap, an approach that combines the major benefits of RADseq (low cost with specific start positions) with those of sequence capture (repeatable sequencing of specific loci) to significantly increase efficiency and reduce costs relative to current approaches. RADcap uses a new version of dual-digest RADseq (3RAD) to identify candidate SNP loci for capture bait design and subsequently uses custom sequence capture baits to consistently enrich candidate SNP loci across many individuals. We combined this approach with a new library preparation method for identifying and removing PCR duplicates from 3RAD libraries, which allows researchers to process RADseq data using traditional pipelines, and we tested the RADcap method by genotyping sets of 96-384 Wisteria plants. Our results demonstrate that our RADcap method: (i) methodologically reduces (to<5%) and allows computational removal of PCR duplicate reads from data, (ii) achieves 80-90% reads on target in 11 of 12 enrichments, (iii) returns consistent coverage (≥4×) across >90% of individuals at up to 99.8% of the targeted loci, (iv) produces consistently high occupancy matrices of genotypes across hundreds of individuals and (v) costs significantly less than current approaches.