Accurate whole-genome sequencing and haplotyping from 10 to 20 human cells.

Recent advances in whole-genome sequencing have brought the vision of personal genomics and genomic medicine closer to reality. However, current methods lack clinical accuracy and the ability to describe the context (haplotypes) in which genome variants co-occur in a cost-effective manner. Here we describe a low-cost DNA sequencing and haplotyping process, long fragment read (LFR) technology, which is similar to sequencing long single DNA molecules without cloning or separation of metaphase chromosomes. In this study, ten LFR libraries were made using only ∼100 picograms of human DNA per sample. Up to 97% of the heterozygous single nucleotide variants were assembled into long haplotype contigs. Removal of false positive single nucleotide variants not phased by multiple LFR haplotypes resulted in a final genome error rate of 1 in 10 megabases. Cost-effective and accurate genome sequencing and haplotyping from 10-20 human cells, as demonstrated here, will enable comprehensive genetic studies and diverse clinical applications.

Bite force estimates in juvenile Tyrannosaurus rex based on simulated puncture marks.

BACKGROUND: Bite marks attributed to adult Tyrannosaurus rex have been subject to numerous studies. However, few bite marks attributed to T. rex have been traced to juveniles, leaving considerable gaps in understanding ontogenetic changes in bite mechanics and force, and the paleoecological role of juvenile tyrannosaurs in the late Cretaceous. METHODS: Here we present bite force estimates for a juvenile Tyrannosaurus rex based on mechanical tests designed to replicate bite marks previously attributed to a T. rex of approximately 13 years old. A maxillary tooth of the juvenile Tyrannosaurus specimen BMR P2002.4.1 was digitized, replicated in dental grade cobalt chromium alloy, and mounted to an electromechanical testing system. The tooth was then pressed into bovine long bones in various locations with differing cortical bone thicknesses at varying speeds for a total of 17 trials. Forces required to replicate punctures were recorded and puncture dimensions were measured. RESULTS: Our experimentally derived linear models suggest bite forces up to 5,641.19 N from cortical bone thickness estimated from puncture marks on an Edmontosaurus and a juvenile Tyrannosaurus. These findings are slightly higher than previously estimated bite forces for a juvenile Tyrannosaurus rex of approximately the same size as BMR P2002.4.1 but fall within the expected range when compared to estimates of adult T. rex. DISCUSSION: The results of this study offer further insight into the role of juvenile tyrannosaurs in late Cretaceous ecosystems. Furthermore, we discuss the implications for feeding mechanisms, feeding behaviors, and ontogenetic niche partitioning.

Feeding traces attributable to juvenile Tyrannosaurus rex offer insight into ontogenetic dietary trends.

Theropod dinosaur feeding traces and tooth marks yield paleobiological and paleoecological implications for social interactions, feeding behaviors, and direct evidence of cannibalism and attempted predation. However, ascertaining the taxonomic origin of a tooth mark is largely dependent on both the known regional biostratigraphy and the ontogenetic stage of the taxon. Currently, most recorded theropod feeding traces and bite marks are attributed to adult theropods, whereas juvenile and subadult tooth marks have been rarely reported in the literature. Here we describe feeding traces attributable to a late-stage juvenile Tyrannosaurus rex on a caudal vertebra of a hadrosaurid dinosaur. The dimensions and spacing of the traces were compared to the dentition of Tyrannosaurus rex maxillae and dentaries of different ontogenetic stages. These comparisons reveal that the tooth marks present on the vertebra closely match the maxillary teeth of a late-stage juvenile Tyrannosaurus rex specimen histologically determined to be 11-12 years of age. These results demonstrate that late-stage juvenile and subadult tyrannosaurs were already utilizing the same large-bodied food sources as adults despite lacking the bone-crushing abilities of adults. Further identification of tyrannosaur feeding traces coupled with experimental studies of the biomechanics of tyrannosaur bite forces from younger ontogenetic stages may reveal dynamic dietary trends and ecological roles of Tyrannosaurus rex throughout ontogeny.

New data towards the development of a comprehensive taphonomic framework for the Late Jurassic Cleveland-Lloyd Dinosaur Quarry, Central Utah.

The Cleveland-Lloyd Dinosaur Quarry (CLDQ) is the densest deposit of Jurassic theropod dinosaurs discovered to date. Unlike typical Jurassic bone deposits, it is dominated by the presence of Allosaurus fragilis. Since excavation began in the 1920s, numerous hypotheses have been put forward to explain the taphonomy of CLDQ, including a predator trap, a drought assemblage, and a poison spring. In an effort to reconcile the various interpretations of the quarry and reach a consensus on the depositional history of CLDQ, new data is required to develop a robust taphonomic framework congruent with all available data. Here we present two new data sets that aid in the development of such a robust taphonomic framework for CLDQ. First, x-ray fluorescence of CLDQ sediments indicate elevated barite and sulfide minerals relative to other sediments from the Morrison Formation in the region, suggesting an ephemeral environment dominated by periods of hypereutrophic conditions during bone accumulation. Second, the degree of abrasion and hydraulic equivalency of small bone fragments dispersed throughout the matrix were analyzed from CLDQ. Results of these analyses suggest that bone fragments are autochthonous or parautochthonous and are derived from bones deposited in the assemblage rather than transported. The variability in abrasion exhibited by the fragments is most parsimoniously explained by local periodic re-working and re-deposition during seasonal fluctuations throughout the duration of the quarry assemblage. Collectively, these data support previous interpretations that the CLDQ represents an attritional assemblage in a poorly-drained overbank deposit where vertebrate remains were introduced post-mortem to an ephemeral pond during flood conditions. Furthermore, while the elevated heavy metals detected at the Cleveland-Lloyd Dinosaur Quarry are not likely the primary driver for the accumulation of carcasses, they are likely the result of multiple sources; some metals may be derived from post-depositional and diagenetic processes, and others are potentially produced from an abundance of decomposing vertebrate carcasses. These new data help to support the inferred depositional environment of the quarry as an ephemeral pond, and represent a significant step in understanding the taphonomy of the bonebed and Late Jurassic paleoecology in this region.

Fluvial transport potential of shed and root-bearing dinosaur teeth from the late Jurassic Morrison Formation.

Shed dinosaur teeth are commonly collected microvertebrate remains that have been used for interpretations of dinosaur feeding behaviors, paleoecology, and population studies. However, such interpretations may be biased by taphonomic processes such as fluvial sorting influenced by tooth shape: shed teeth, removed from the skull during life, and teeth possessing roots, removed from the skull after death. As such, teeth may behave differently in fluvial systems due to their differences in shape. In order to determine the influence of fluvial processes on the preservation and distribution of shed and root-bearing dinosaur teeth, the hydrodynamic behaviors of high-density urethane resin casts of shed and root-bearing Allosaurus and Camarasaurus teeth were experimentally tested for relative transport distances at increasing flow velocities in an artificial fluviatile environment. Results show that tooth cast specimens exhibited comparable patterns of transport at lower velocities, though the shed Camarasaurus teeth transported considerably farther in medium to higher flow velocities. Two-Way ANOVA tests indicate significant differences in the mean transport distances of tooth casts oriented perpendicular to flow (p < 0.05) with varying tooth morphologies and flow velocities. The differences exhibited in the transportability of shed and root-bearing teeth has important implications for taphonomic reconstructions, as well as future studies on dinosaur population dynamics, paleoecology, and feeding behaviors.

Distributions of cranial pathologies provide evidence for head-butting in dome-headed dinosaurs (Pachycephalosauridae).

Pachycephalosaurids are small, herbivorous dinosaurs with domed skulls formed by massive thickening of the cranial roof. The function of the dome has been a focus of debate: the dome has variously been interpreted as the product of sexual selection, as an adaptation for species recognition, or as a weapon employed in intraspecific combat, where it was used in butting matches as in extant ungulates. This last hypothesis is supported by the recent identification of cranial pathologies in pachycephalosaurids, which appear to represent infections resulting from trauma. However, the frequency and distribution of pathologies have not been studied in a systematic fashion. Here, we show that pachycephalosaurids are characterized by a remarkably high incidence of cranial injury, where 22% of specimens have lesions on the dome. Frequency of injury shows no significant difference between different genera, but flat-headed morphs (here interpreted as juveniles or females) lack lesions. Mapping of injuries onto a digitial pachycephalosaurid skull shows that although lesions are distributed across the dome, they cluster near the apex, which is consistent with the hypothesis that the dome functioned for intraspecific butting matches.

Cranial pathologies in a specimen of Pachycephalosaurus.

BACKGROUND: A frontoparietal dome of a large pachycephalosaurid collected from the Upper Cretaceous Hell Creek Formation in 2001 is identified as Pachycephalosaurus wyomingensis. The specimen features two large oval depressions on the dorsal surface, accompanied by numerous circular pits on the margin and inner surface of the larger depressions. METHODOLOGY/PRINCIPAL FINDINGS: In order to identify the origin of these structures, computed tomography (CT) data and morphological characteristics of the specimen are analyzed and compared with similar osteological structures in fossil and extant archosaurs caused by taphonomic processes, non-pathologic bone resorption, and traumatic infection/inflammatory origins. The results of these analyses suggest that the structures are pathologic lesions likely resulting from a traumatic injury and followed by secondary infection at the site. CONCLUSIONS/SIGNIFICANCE: The presence of lesions on a frontoparietal dome, and the exclusivity of their distribution along the dorsal dome surface, offers further insight into frontoparietal dome function and supports previously hypothesized agonistic behavior in pachycephalosaurids.

Influence of microbial biofilms on the preservation of primary soft tissue in fossil and extant archosaurs.

BACKGROUND: Mineralized and permineralized bone is the most common form of fossilization in the vertebrate record. Preservation of gross soft tissues is extremely rare, but recent studies have suggested that primary soft tissues and biomolecules are more commonly preserved within preserved bones than had been presumed. Some of these claims have been challenged, with presentation of evidence suggesting that some of the structures are microbial artifacts, not primary soft tissues. The identification of biomolecules in fossil vertebrate extracts from a specimen of Brachylophosaurus canadensis has shown the interpretation of preserved organic remains as microbial biofilm to be highly unlikely. These discussions also propose a variety of potential mechanisms that would permit the preservation of soft-tissues in vertebrate fossils over geologic time. METHODOLOGY/PRINCIPAL FINDINGS: This study experimentally examines the role of microbial biofilms in soft-tissue preservation in vertebrate fossils by quantitatively establishing the growth and morphology of biofilms on extant archosaur bone. These results are microscopically and morphologically compared with soft-tissue extracts from vertebrate fossils from the Hell Creek Formation of southeastern Montana (Latest Maastrichtian) in order to investigate the potential role of microbial biofilms on the preservation of fossil bone and bound organic matter in a variety of taphonomic settings. Based on these analyses, we highlight a mechanism whereby this bound organic matter may be preserved. CONCLUSIONS/SIGNIFICANCE: Results of the study indicate that the crystallization of microbial biofilms on decomposing organic matter within vertebrate bone in early taphonomic stages may contribute to the preservation of primary soft tissues deeper in the bone structure.

A modified technique for anterior multilevel cervical fusion.

Anterior cervical fusion with interbody bone graft and anterior plating is commonly performed. Unfortunately, the plate has been reported to shield the graft from loading, thus reducing fusion rates. Interbody fusion cages have been effective in the lumbar spine and have gained acceptance in the cervical spine. Twenty-five patients underwent anterior cervical fusion with this modified technique. All patients received anterior diskectomy and corpectomy, placement of an interbody fusion cage packed with corpectomy bone, and application of an anterior cervical plate. Fusion was defined by radiographic evidence of trabecular bone bridging through the cage. No external bracing was used except soft collars as needed. Pre- and postoperative pain scales were completed and statistically analyzed using paired t tests. There were no cases of pseudoarthrosis or major neurological, vascular, or wound complications. There was one case of mild dysphagia that remained unresolved. Mean operative time was comparable to standard instrumented multilevel cervical fusion surgeries. Visual analogue pain scales were significantly improved following surgery. The advantages of using interbody cages with anterior plating include immediate stability and support, elimination of donor site pain from iliac crest bone autograft, and a decrease in pseudoarthrosis by halving the number of fusion surfaces.