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Infections by several DNA viruses can severely impact outcomes in paediatric immunocompromised patients. Current testing, which is generally limited to singleplex qPCR assays, can miss both common and rarer viruses if they are not targeted.To evaluate the performance of the Galileo Viral Panel (Galileo), a sample-to-result shotgun metagenomics platform for the detection and quantification of 12 DNA viruses, compared to standard of care qPCR assays.A clinical performance evaluation was carried out using 43 prospectively collected EDTA plasma samples positive for one or more DNA viruses. Agreement between assays was assessed by overall, positive, and negative percent agreement, as well as quantitative agreement by linear regression and Bland-Altman analysis.Overall positive percent agreement was 84% (95% CI: 76%-90%), and negative percent agreement was 95% (95% CI: 92%-97%). There was a high correlation between Galileo and qPCR for ADV, CMV, EBV, and VZV (We observed high qualitative and quantitative agreement between qPCR and Galileo. Galileo identified additional viruses that were not tested with routine qPCR and could impact clinical outcomes.

Genetic markers are important resources for individual identification and parentage assessment. Although short tandem repeats (STRs) have been the traditional DNA marker, technological advances have led to single nucleotide polymorphisms (SNPs) becoming an attractive alternative. SNPs can be highly multiplexed and automatically scored, which allows for easier standardization and sharing among laboratories. Equine parentage is currently assessed using STRs. We obtained a publicly available SNP dataset of 729 horses representing 32 diverse breeds. A proposed set of 101 SNPs was analyzed for DNA typing suitability. The overall minor allele frequency of the panel was 0.376 (range 0.304-0.419), with per breed probability of identities ranging from 5.6 × 10-35 to 1.86 × 10-42 . When one parent was available, exclusion probabilities ranged from 0.9998 to 0.999996, although when both parents were available, all breeds had exclusion probabilities greater than 0.9999999. A set of 388 horses from 35 breeds was genotyped to evaluate marker performance on known families. The set included 107 parent-offspring pairs and 101 full trios. No horses shared identical genotypes across all markers, indicating that the selected set was sufficient for individual identification. All pairwise comparisons were classified using ISAG rules, with one or two excluding markers considered an accepted parent-offspring pair, two or three excluding markers considered doubtful and four or more excluding markers rejecting parentage. The panel had an overall accuracy of 99.9% for identifying true parent-offspring pairs. Our developed marker set is both present on current generation SNP chips and can be highly multiplexed in standalone panels and thus is a promising resource for SNP-based DNA typing.

Infections with DNA viruses are frequent causes of morbidity and mortality in transplant recipients., Infections with DNA viruses are frequent causes of morbidity and mortality in transplant recipients. This study describes the analytical and clinical performance characteristics of the Arc Bio Galileo Pathogen Solution, an all-inclusive metagenomic next-generation sequencing (mNGS) reagent and bioinformatics pipeline that allows the simultaneous quantitation of 10 transplant-related double-stranded DNA (dsDNA) viruses (adenovirus [ADV], BK virus [BKV], cytomegalovirus [CMV], Epstein-Barr virus [EBV], human herpesvirus 6A [HHV-6A], HHV-6B, herpes simplex virus 1 [HSV-1], HSV-2, JC virus [JCV], and varicella-zoster virus [VZV]). The mNGS 95% limit of detection ranged from 14 copies/ml (HHV-6) to 191 copies/ml (BKV), and the lower limit of quantitation ranged from 442 international units (IU)/ml (EBV) to 661 copies/ml (VZV). An evaluation of 50 residual plasma samples with at least one DNA virus detected in prior clinical testing showed a total percent agreement of mNGS and quantitative PCR (qPCR) of 89.2% (306/343), with a κ statistic of 0.725. The positive percent agreement was 84.9% (73/86), and the negative percent agreement was 90.7% (233/257). Furthermore, mNGS detected seven subsequently confirmed coinfections that were not initially requested by qPCR. Passing-Bablok regression revealed a regression line of y = 0.953x + 0.075 (95% confidence interval [CI] of the slope, 0.883 to 1.011; intercept, −0.100 to 0.299), and Bland-Altman analysis (mNGS − qPCR) showed a slight positive bias (0.28 log10 concentration; 95% limits of agreement, −0.62 to 1.18). In conclusion, the mNGS-based Galileo pipeline demonstrates analytical and clinical performance comparable to that of qPCR for transplant-related DNA viruses.

SUMMARYSLC45A2 mutations are responsible for several dilution phenotypes in horses, including cream, pearl, and sunshine. We sequenced the SLC45A2 gene in a horse that possessed a diluted coat color but tested negative for any known dilution genotypes. We identified a novel homozygous missense mutation in ECA21: SLC45A2:c.305G>A; p.(Arg102Gln) which we have named snowdrop (Csno). The snowdrop dilution is autosomal recessive and dilutes both red and black pigment in the homozygous state, creating a phenotype similar to homozygous cream. We also identified this mutation in the heterozygous state in several relatives, including the sire and dam of the affected horse, where it has no visible effect on phenotype. Based on these data, the snowdrop variant produces a recessive dilution similar to cream, and its discovery confirms that multiple SLC45A2 alleles cause dilute phenotypes in horses.

Between 1500 and 1850, more than 12 million enslaved Africans were transported to the New World. The vast majority were shipped from West and West-Central Africa, but their precise origins are largely unknown. We used genome-wide ancient DNA analyses to investigate the genetic origins of three enslaved Africans whose remains were recovered on the Caribbean island of Saint Martin. We trace their origins to distinct subcontinental source populations within Africa, including Bantu-speaking groups from northern Cameroon and non-Bantu speakers living in present-day Nigeria and Ghana. To our knowledge, these findings provide the first direct evidence for the ethnic origins of enslaved Africans, at a time for which historical records are scarce, and demonstrate that genomic data provide another type of record that can shed new light on long-standing historical questions.

Patagonia was the last region of the Americas reached by humans who entered the continent from Siberia ∼15,000–20,000 y ago. Despite recent genomic approaches to reconstruct the continental evolutionary history, regional characterization of ancient and modern genomes remains understudied. Exploring the genomic diversity within Patagonia is not just a valuable strategy to gain a better understanding of the history and diversification of human populations in the southernmost tip of the Americas, but it would also improve the representation of Native American diversity in global databases of human variation. Here, we present genome data from four modern populations from Central Southern Chile and Patagonia (n = 61) and four ancient maritime individuals from Patagonia (∼1,000 y old). Both the modern and ancient individuals studied in this work have a greater genetic affinity with other modern Native Americans than to any non-American population, showing within South America a clear structure between major geographical regions. Native Patagonian Kawéskar and Yámana showed the highest genetic affinity with the ancient individuals, indicating genetic continuity in the region during the past 1,000 y before present, together with an important agreement between the ethnic affiliation and historical distribution of both groups. Lastly, the ancient maritime individuals were genetically equidistant to a ∼200-y-old terrestrial hunter-gatherer from Tierra del Fuego, which supports a model with an initial separation of a common ancestral group to both maritime populations from a terrestrial population, with a later diversification of the maritime groups. © 2018 National Academy of Sciences. All Rights Reserved. aHuman Genetics Program, Institute of Biomedical Sciences, Faculty of Medicine, University of Chile, Santiago 8380453, Chile; bCentre for GeoGenetics, University of Copenhagen, 1350 Copenhagen, Denmark; cInternational Laboratory for Human Genome Research, National Autonomous University of Mexico, Juriquilla 76230, Santiago de Querétaro, Mexico; dCenter for Computational, Evolutionary and Human Genomics, Stanford University, Stanford, CA 94305; eArc Bio, LLC, Menlo Park, CA 94025; fDepartment of Genetics, Stanford University, Stanford, CA 94305; gInstitute of Ecology and Evolution, University of Bern, 3012 Bern, Switzerland; hCentro de Estudios del Hombre Austral, Instituto de la Patagonia, Universidad de Magallanes, Punta Arenas 6213029, Chile; iNational Laboratory of Genomics for Biodiversity, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Irapuato, 36821 Guanajuato, Mexico; jInterdisciplinary Centre of Marine and Environmental Research, University of Porto, Novo Edificio do Terminal de Cruzeiros do Porto de Leixões, 4450-208 Matosinhos, Portugal; kDepartment of Medicine, University of California, San Francisco, CA 94131; lDepartment of Bioengineering and Therapeutic Science, University of California, San Francisco, CA 94158; mDepartment of Computational Biology, University of Lausanne, 1015 Lausanne, Switzerland; nDepartment of Zoology, University of Cambridge, Cambridge CB2 1TN, United Kingdom; and oWellcome Genome Campus, Wellcome Trust Sanger Institute, Hinxton CB10 1SA, United Kingdom ACKNOWLEDGMENTS. We thank the contribution of the “Instituto de la Patagonia” and Stanford Sequencing Center. We thank Consuelo Quinto for helpful comments on earlier versions of this manuscript. This work was supported by Comisión Nacional de Investigación Científica y Tecnológica Grant USA2013-0015 and Fondo Nacional de Ciencia y Tecnología Grants 1140544 and 1170726. M.C.Á.-A.’s laboratory is supported by Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica–Universidad Nacional Autónoma de México Grant IA206817. A.-S.M. and D.C.D. were supported by Swiss National Science Foundation Grant PZ00P3_154717 and European Research Council Starting Grant 679330. A.M.-E. was supported by International Center for Genetic Engineering and Biotechnology Grant CRP/ MEX15-04_EC. Sequencing of modern DNA was partially funded by Fund of Scientific and Technological Equipment (FONDEQUIP) Grant EQM140157.

SummaryGenome sequences of diverse free-living protists are essential for understanding eukaryotic evolution and molecular and cell biology. The free-living amoeboflagellate Naegleria gruberi belongs to a varied and ubiquitous protist clade (Heterolobosea) that diverged from other eukaryotic lineages over a billion years ago. Analysis of the 15,727 protein-coding genes encoded by Naegleria's 41 Mb nuclear genome indicates a capacity for both aerobic respiration and anaerobic metabolism with concomitant hydrogen production, with fundamental implications for the evolution of organelle metabolism. The Naegleria genome facilitates substantially broader phylogenomic comparisons of free-living eukaryotes than previously possible, allowing us to identify thousands of genes likely present in the pan-eukaryotic ancestor, with 40% likely eukaryotic inventions. Moreover, we construct a comprehensive catalog of amoeboid-motility genes. The Naegleria genome, analyzed in the context of other protists, reveals a remarkably complex ancestral eukaryote with a rich repertoire of cytoskeletal, sexual, signaling, and metabolic modules.PaperClip

Placozoans are arguably the simplest free-living animals, possibly evoking an early stage in metazoan evolution, yet their biology is poorly understood. Here we report the sequencing and analysis of the {approx}98 million base pair nuclear genome of the placozoan Trichoplax adhaerens. Whole genome phylogenetic analysis suggests that placozoans belong to a 'eumetazoan' clade that includes cnidarians and bilaterians, with sponges as the earliest diverging animals. The compact genome exhibits conserved gene content, gene structure, and synteny relative to the human and other complex eumetazoan genomes. Despite the apparent cellular and organismal simplicity of Trichoplax, its genome encodes a rich array of transcription factor and signaling pathway genes that are typically associated with diverse cell types and developmental processes in eumetazoans, motivating further searches for cryptic cellular complexity and/or as yet unobserved life history stages.

The diplomonad parasite Giardia intestinalis contains two functionally equivalent nuclei that are inherited independently during mitosis. Although presumed to be asexual, Giardia has low levels of allelic heterozygosity, indicating that the two nuclear genomes may exchange genetic material. Fluorescence in situ hybridization performed with probes to an episomal plasmid suggests that plasmids are transferred between nuclei in the cyst, and transmission electron micrographs demonstrate fusion between cyst nuclei. Green fluorescent protein fusions of giardial homologs of meiosis-specific genes localized to the nuclei of cysts, but not the vegetative trophozoite. These data suggest that the fusion of nuclei, or karyogamy, and subsequently somatic homologous recombination facilitated by the meiosis gene homologs, occur in the giardial cyst.

Summary 1. Application of whole-genome capture (WGC) methods to ancient DNA (aDNA) promises to increase efficiency of ancient genome sequencing. 2. We compared the performance of two recent WGC methods in enriching human aDNA within Illumina libraries built using both double-stranded and single-stranded build protocols. Although both methods effectively enriched aDNA, we observed consistent differences between the methods, providing the opportunity to further explore parameters influencing WGC experiments. 3. Our results suggest bait length has a potential influence on library enrichment. Moreover, we show WGC biases against shorter molecules that are enriched in single-stranded libraries preparation protocols. Lastly, we document the effect of WGC on features including clonality, GC composition and repetitive DNA content. 4. Our findings provide insights relevant to those planning WGC on aDNA and suggest future tests and optimization to improve WGC efficiency.

1. The application of whole genome capture (WGC) methods to ancient DNA (aDNA) promises to increase the efficiency of ancient genome sequencing. 2. We compared the performance of two recently developed WGC methods in enriching human aDNA within Illumina libraries built using both double-stranded (DSL) and single-stranded (SSL) build protocols. Although both methods effectively enriched aDNA, one consistently produced marginally better results, giving us the opportunity to further explore the parameters influencing WGC experiments. 3. Our results suggest that bait length has an important influence on library enrichment. Moreover, we show that WGC biases against the shorter molecules that are enriched in SSL preparation protocols. Therefore application of WGC to such samples is not recommended without future optimization. Lastly, we document the effect of WGC on other features including clonality, GC composition and repetitive DNA content of captured libraries. 4. Our findings provide insights for researchers planning to perform WGC on aDNA, and suggest future tests and optimization to improve WGC efficiency.

Sikora, Martin et al., Genome sequencing of the 5,300-year-old mummy of the Tyrolean Iceman, found in 1991 on a glacier near the border of Italy and Austria, has yielded new insights into his origin and relationship to modern European populations. A key finding of that study was an apparent recent common ancestry with individuals from Sardinia, based largely on the Y chromosome haplogroup and common autosomal SNP variation. Here, we compiled and analyzed genomic datasets from both modern and ancient Europeans, including genome sequence data from over 400 Sardinians and two ancient Thracians from Bulgaria, to investigate this result in greater detail and determine its implications for the genetic structure of Neolithic Europe. Using whole-genome sequencing data, we confirm that the Iceman is, indeed, most closely related to Sardinians. Furthermore, we show that this relationship extends to other individuals from cultural contexts associated with the spread of agriculture during the Neolithic transition, in contrast to individuals from a hunter-gatherer context. We hypothesize that this genetic affinity of ancient samples from different parts of Europe with Sardinians represents a common genetic component that was geographically widespread across Europe during the Neolithic, likely related to migrations and population expansions associated with the spread of agriculture. © 2014 Sikora et al., This project was supported by NIH grant 2R01HG003229. This work was also partially supported by an Erwin Schrödinger Fellowship of the Austrian Science Fund (FWF) to MS (J3375-B19).

Most ancient specimens contain very low levels of endogenous DNA, precluding the shotgun sequencing of many interesting samples because of cost. Ancient DNA (aDNA) libraries often contain

The protozoan parasite Giardia intestinalis (also known as Giardia lamblia) is a major waterborne pathogen. During its life cycle, Giardia alternates between the actively growing trophozoite, which has two diploid nuclei with low levels of allelic heterozygosity, and the infectious cyst, which has four nuclei and a tough outer wall. Although the formation of the cyst wall has been studied extensively, we still lack basic knowledge about many fundamental aspects of the cyst, including the sources of the four nuclei and their distribution during the transformation from cyst into trophozoite. In this study, we tracked the identities of the nuclei in the trophozoite and cyst using integrated nuclear markers and immunofluorescence staining. We demonstrate that the cyst is formed from a single trophozoite by a mitotic division without cytokinesis and not by the fusion of two trophozoites. During excystation, the cell completes cytokinesis to form two daughter trophozoites. The non-identical nuclear pairs derived from the parent trophozoite remain associated in the cyst and are distributed to daughter cells during excystation as pairs. Thus, nuclear sorting (such that each daughter cell receives a pair of identical nuclei) does not appear to be a mechanism by which Giardia reduces heterozygosity between its nuclei. Rather, we show that the cyst nuclei exchange chromosomal genetic material, perhaps as a way to reduce heterozygosity in the absence of meiosis and sex, which have not been described in Giardia. These results shed light on fundamental aspects of the Giardia life cycle and have implications for our understanding of the population genetics and cell biology of this binucleate parasite.

We used translation-blocking morpholinos to reduce protein levels in Giardia intestinalis . Twenty-four hours after electroporation with morpholinos targeting either green fluorescent protein or kinesin-2b, levels of these proteins were reduced by 60%. An epitope-tagged transgene can also be used as a reporter for morpholino efficacy with targets lacking specific antibodies.

Background Targeted capture of genomic regions reduces sequencing cost while generating higher coverage by allowing biomedical researchers to focus on specific loci of interest, such as exons. Targeted capture also has the potential to facilitate the generation of genomic data from DNA collected via saliva or buccal cells. DNA samples derived from these cell types tend to have a lower human DNA yield, may be degraded from age and/or have contamination from bacteria or other ambient oral microbiota. However, thousands of samples have been previously collected from these cell types, and saliva collection has the advantage that it is a non-invasive and appropriate for a wide variety of research. Results We demonstrate successful enrichment and sequencing of 15 South African KhoeSan exomes and 2 full genomes with samples initially derived from saliva. The expanded exome dataset enables us to characterize genetic diversity free from ascertainment bias for multiple KhoeSan populations, including new exome data from six HGDP Namibian San, revealing substantial population structure across the Kalahari Desert region. Additionally, we discover and independently verify thirty-one previously unknown KIR alleles using methods we developed to accurately map and call the highly polymorphic HLA and KIR loci from exome capture data. Finally, we show that exome capture of saliva-derived DNA yields sufficient non-human sequences to characterize oral microbial communities, including detection of bacteria linked to oral disease (e.g. Prevotella melaninogenica). For comparison, two samples were sequenced using standard full genome library preparation without exome capture and we found no systematic bias of metagenomic information between exome-captured and non-captured data. Conclusions DNA from human saliva samples, collected and extracted using standard procedures, can be used to successfully sequence high quality human exomes, and metagenomic data can be derived from non-human reads. We find that individuals from the Kalahari carry a higher oral pathogenic microbial load than samples surveyed in the Human Microbiome Project. Additionally, rare variants present in the exomes suggest strong population structure across different KhoeSan populations.