High throughput sequencing (HTS) has been used for a number of years in the field of paleogenomics to facilitate the recovery of small DNA fragments from ancient specimens. Recently, these techniques have also been applied in forensics, where they have been used for the recovery of mitochondrial DNA sequences from samples where traditional PCR-based assays fail because of the very short length of endogenous DNA molecules. Here, we describe the biological sexing of a ~4000-year-old Egyptian mummy using shotgun sequencing and two established methods of biological sex determination (RX and RY), by way of mitochondrial genome analysis as a means of sequence data authentication. This particular case of historical interest increases the potential utility of HTS techniques for forensic purposes by demonstrating that data from the more discriminatory nuclear genome can be recovered from the most damaged specimens, even in cases where mitochondrial DNA cannot be recovered with current PCR-based forensic technologies. Although additional work remains to be done before nuclear DNA recovered via these methods can be used routinely in operational casework for individual identification purposes, these results indicate substantial promise for the retrieval of probative individually identifying DNA data from the most limited and degraded forensic specimens.

Premise of the Study The development of pipelines for locus discovery has spurred the use of target enrichment for plant phylogenomics. However, few studies have compared pipelines from locus discovery and bait design, through validation, to tree inference. We compared three methods within Leguminosae (Fabaceae) and present a workflow for future efforts. Methods Using 30 transcriptomes, we compared Hyb-Seq, MarkerMiner, and the Yang and Smith (Y&S) pipelines for locus discovery, validated 7501 baits targeting 507 loci across 25 genera via Illumina sequencing, and inferred gene and species trees via concatenation- and coalescent-based methods. Results Hyb-Seq discovered loci with the longest mean length. MarkerMiner discovered the most conserved loci with the least flagged as paralogous. Y&S offered the most parsimony-informative sites and putative orthologs. Target recovery averaged 93% across taxa. We optimized our targeted locus set based on a workflow designed to minimize paralog/ortholog conflation and thus present 423 loci for legume phylogenomics. Conclusions Methods differed across criteria important for phylogenetic marker development. We recommend Hyb-Seq as a method that may be useful for most phylogenomic projects. Our targeted locus set is a resource for future, community-driven efforts to reconstruct the legume tree of life.

The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method, we incorporated an internal GFP cleavage control to assess the extent of library cleavage as well as Sanger sequencing of the cleaved library to assess PAM depletion prior to next-generation sequencing. We also detail the methods needed to construct all relevant DNA constructs, and how to troubleshoot the assay. We finally demonstrate the technique by determining PAM sequences recognized by the Neisseria meningitidis Cas9, revealing subtle sequence requirements of this highly specific PAM. The overall method offers a rapid means to identify PAMs recognized by diverse CRISPR nucleases, with the potential to greatly accelerate our ability to characterize and harness novel CRISPR nucleases across their many uses.

The Caribbean was one of the last parts of the Americas to be settled by humans, but how and when the islands were first occupied remains a matter of debate. Ancient DNA can help answering these questions, but the work has been hampered by poor DNA preservation. We report the genome sequence of a 1,000-year-old Lucayan Taino individual recovered from the site of Preacher’s Cave in the Bahamas. We sequenced her genome to 12.4-fold coverage and show that she is genetically most closely related to present-day Arawakan speakers from northern South America, suggesting that the ancestors of the Lucayans originated there. Further, we find no evidence for recent inbreeding or isolation in the ancient genome, suggesting that the Lucayans had a relatively large effective population size. Finally, we show that the native American components in some present-day Caribbean genomes are closely related to the ancient Taino, demonstrating an element of continuity between precontact populations and present-day Latino populations in the Caribbean.

We resolve a longstanding question regarding the kinship of two high-status Egyptians from the 12th Dynasty, Nakht-Ankh and Khnum-Nakht, whose mummies were discovered in 1907 by Egyptian workmen directed by Flinders Petrie and Ernest Mackay. Although their coffin inscriptions indicate that Nakht-Ankh and Khnum-Nakht were brothers, when the mummies were unwrapped in 1908 the skeletal morphologies were found to be quite different, suggesting an absence of family relationship. We extracted ancient DNA from the teeth of the two mummies and, following hybridization capture of the mitochondrial and Y chromosome fractions, sequenced the DNA by a next generation method. Analysis of single nucleotide polymorphisms showed that both Nakht-Ankh and Khnum-Nakht belonged to mitochondrial haplotype M1a1, suggesting a maternal relationship. The Y chromosome sequences were less complete but showed variations between the two mummies, indicating that Nakht-Ankh and Khnum-Nakht had different fathers. Our study emphasizes the importance of kinship in ancient Egypt, and represents the first successful typing of both mitochondrial and Y chromosomal DNA in Egyptian mummies.

Life cycle strategies have evolved extensively throughout the history of metazoans. The expression of disparate life stages within a single ontogeny can present conflicts to trait evolution, and therefore may have played a major role in shaping metazoan forms. However, few studies have examined the consequences of adding or subtracting life stages on patterns of trait evolution. By analysing trait evolution in a clade of closely related salamander lineages we show that shifts in the number of life cycle stages are associated with rapid phenotypic evolution. Specifically, salamanders with an aquatic-only (paedomorphic) life cycle have frequently added vertebrae to their trunk skeleton compared with closely related lineages with a complex aquatic-to-terrestrial (biphasic) life cycle. The rate of vertebral column evolution is also substantially lower in biphasic lineages, which may reflect the functional compromise of a complex cycle. This study demonstrates that the consequences of life cycle evolution can be detected at very fine scales of divergence. Rapid evolutionary responses can result from shifts in selective regimes following changes in life cycle complexity.

Author summary Mycobacterium leprae, which causes leprosy in humans, also infects nine-banded armadillos, red squirrels, and nonhuman primates. Genomic data for M. leprae strains from wild armadillos and red squirrels show that humans were responsible for the original introduction of M. leprae to these species. It is not known whether naturally occurring leprosy among nonhuman primates is due to incidental infections from humans or whether nonhuman primates can serve as a host for M. leprae. To this end, we sequenced complete genomes of M. leprae strains from three naturally infected nonhuman primates. Our results suggest that M. leprae strains can be transmitted from humans to nonhuman primates as well as between nonhuman primate species, and thus, other primates might serve as a host for M. leprae in the wild. We also assessed whether wild ring-tailed lemurs from Madagascar and chimpanzees from Uganda showed presence of M. leprae infection. Although these populations tested negative for M. leprae infection, further research on the prevalence of M. leprae in other wild nonhuman primate populations, especially in leprosy-endemic regions, is warranted.