Summary

Background

Direct whole-genome sequencing of Mycobacterium tuberculosis from clinical specimens will be a major breakthrough in tuberculosis diagnosis and control. To date, direct whole-genome sequencing has never been used in genomic epidemiology, and its accuracy in transmission inference remains unknown. We investigated the technical challenges imposed by direct whole-genome sequencing, and used it to infer transmission clusters and predict drug resistance.

Methods

Using an optimised workflow, we did direct whole-genome sequencing for 37 clinical specimens from 23 tuberculosis patients. Nine sputum samples from nine patients who were infected with different non-tuberculous mycobacteria and culture-negative for tuberculosis were used as controls in the qPCR assays and pre-sequencing runs. Additionally, 780 clinical isolates in the region of Comunidad Valenciana (Spain) were whole-genome sequenced between Jan 1, 2014, and Dec 31, 2016. We analysed the genomic variants to build a tuberculosis transmission network for the region, including the clinical specimens, and to predict drug susceptibility profiles.

Findings

After sequencing 37 clinical specimens, 28 specimens (22 [85%] of 26 smear-positive and six [55%] of 11 smear-negative) met the quality criteria for downstream analysis. All 28 clinical specimens clustered with their matching culture isolates, with a median distance of 0 single nucleotide polymorphisms. Of the 28 clinical specimens, 16 (57%) were accurately assigned to ten transmission clusters in the region, and 12 (43%) were unique cases. Transmission inferences and drug-susceptibility predictions from direct whole-genome sequencing data were concordant with sequences from corresponding cultures and phenotypic drug-susceptibility testing. Complete genomic analysis, within a week of specimen receipt, cost €217 per sample (excluding personnel costs).

Interpretation

Direct whole-genome sequencing could be used to accurately delineate transmission clusters of tuberculosis and conduct culture-independent surveillance. Compared with conventional approaches, direct whole-genome sequencing allows researchers to do real-time genomic epidemiology and drug resistance surveillance in settings where culture and drug susceptibility testing are not available.

Funding

European Research Council; Ministerio de Ciencia, Innovación y Universidades (Spanish Government).

The phylogenetic relationships for Ipomoea species are incongruent in previous studies. Comparative karyotype analysis can provide valuable information for phylogenetic relationships among species. A reliable and efficient system for chromosome identification is the foundation for karyotype analysis. However, for the Ipomoea species, individual chromosomes were not identified in all previous reported karyotypes due to their small size, high number and similar morphology. Fluorescence in situ hybridization (FISH) using oligonucleotides (oligos) as probes is a new strategy for chromosome identification and karyotype analysis. Here, we developed the first set of oligo-based probes based on the reference genome of Ipomoea nil, a model species in the genus Ipomoea. In all, we developed four oligo-FISH probes. By a combined use of four oligo probes, sequential FISH analysis were conducted on the same metaphase cells with each round including two probes, which permitted simultaneous identification of all I. nil chromosomes and anchoring 15 pseudochromosomes to individual cytological chromosomes. Moreover, 45S and 5S rDNA were mapped to specific chromosomes. A karyotype based on individually identified chromosomes was established, which was the first FISH-based molecular cytogenetic karyotype of I. nil. Our study has created the basis for studying chromosome variation and evolution in genus Ipomoea by comparative karyotype analysis using this set of oligo probes.

The cave lion is an extinct felid that was widespread across the Holarctic throughout the Late Pleistocene. Its closest extant relative is the lion (Panthera leo), but the timing of the divergence between these two taxa, as well as their taxonomic ranking are contentious. In this study we analyse 31 mitochondrial genome sequences from cave lion individuals that, through a combination of 14C and genetic tip dating, are estimated to be from dates extending well into the mid-Pleistocene. We identified two deeply diverged and well-supported reciprocally monophyletic mitogenome clades in the cave lion, and an additional third distinct lineage represented by a single individual. One of these clades was restricted to Beringia while the other was prevalent across western Eurasia. These observed clade distributions are in line with previous observations that Beringian and European cave lions were morphologically distinct. The divergence dates for these lineages are estimated to be far older than those between extant lions subspecies. By combining our radiocarbon tip-dates with a split time prior that takes into account the most up-to-date fossil stem calibrations, we estimated the mitochondrial DNA divergence between cave lions and lions to be 1.85 Million ya (95% 0.52– 2.91 Mya). Taken together, these results support previous hypotheses that cave lions existed as at least two subspecies during the Pleistocene, and that lions and cave lions were distinct species.

Abstract Relationships of the Neotropical finches in the genera Euphonia and Chlorophonia (Fringillidae: Euphoniinae) have been clarified by recent molecular studies, but species-level relationships within this group have not been thoroughly addressed. In this study, we sampled specimens representing every recognized species of these genera, in addition to 2 outgroup taxa, and used target enrichment to sequence thousands of ultraconserved element (UCE) loci, as well as mitochondrial DNA reconstructed from off-target reads, from each specimen to infer these relationships. We constructed both concatenation and coalescent-based estimates of phylogeny from this dataset using matrices of varying levels of completeness, and we generated a time-scaled ultrametric tree using a recently published fossil-based external calibration. We found uniformly strong support for a monophyletic subfamily Euphoniinae and genus Chlorophonia, but a paraphyletic Euphonia across UCEs and mitochondrial genomes. Otherwise, our inferred relationships were largely concordant with previous studies. Our time-tree indicated a stem divergence time of 13.8 million years ago for this lineage, followed by a relatively young crown age of only 7.1 myr. Reconstructions of biogeographic history based on this tree suggest a South American origin for crown Euphoniinae, possibly resulting from a transoceanic dispersal event from the Eastern Hemisphere, followed by 2 dispersal events into the Caribbean and as many as 6 invasions of North America coinciding with recent estimates of the age at which the Isthmus of Panama had completely formed. We recommend splitting Euphonia and resurrecting the genus Cyanophonia for the 3 blue-hooded species more closely related to Chlorophonia. Based on our results, we suspect that there is undescribed species-level diversity in at least one, possibly many, widespread and phenotypically diverse species.

Vaccination has transformed public health, most notably including the eradication of smallpox. Despite its profound historical importance, little is known of the origins and diversity of the viruses used in smallpox vaccination. Prior to the twentieth century, the method, source and origin of smallpox vaccinations remained unstandardised and opaque. We reconstruct and analyse viral vaccine genomes associated with smallpox vaccination from historical artefacts. Significantly, we recover viral molecules through non-destructive sampling of historical materials lacking signs of biological residues. We use the authenticated ancient genomes to reveal the evolutionary relationships of smallpox vaccination viruses within the poxviruses as a whole.

Polycomb group (PcG) proteins silence gene expression by chemically and physically modifying chromatin. A subset of PcG target loci are compacted and cluster in the nucleus; a conformation that is thought to contribute to gene silencing. However, how these interactions influence gross nuclear organization and their relationship with transcription remains poorly understood. Here we examine the role of Polycomb-repressive complex 1 (PRC1) in shaping 3D genome organization in mouse embryonic stem cells (mESCs). Using a combination of imaging and Hi-C analyses, we show that PRC1-mediated long-range interactions are independent of CTCF and can bridge sites at a megabase scale. Impairment of PRC1 enzymatic activity does not directly disrupt these interactions. We demonstrate that PcG targets coalesce in vivo, and that developmentally induced expression of one of the target loci disrupts this spatial arrangement. Finally, we show that transcriptional activation and the loss of PRC1-mediated interactions are separable events. These findings provide important insights into the function of PRC1, while highlighting the complexity of this regulatory system.

Research on the ecology and evolution of viruses is often hampered by the limitation of sequence information to short parts of the genomes or single genomes derived from cultures. In this study, we use hybrid sequence capture enrichment in combination with high-throughput sequencing to provide efficient access to full genomes of European hantaviruses from rodent samples obtained in the field. We applied this methodology to Tula (TULV) and Puumala (PUUV) orthohantaviruses for which analyses from natural host samples are typically restricted to partial sequences of their tri-segmented RNA genome. We assembled a total of ten novel hantavirus genomes de novo with very high coverage (on average >99%) and sequencing depth (average >247×). A comparison with partial Sanger sequences indicated an accuracy of >99.9% for the assemblies. An analysis of two common vole (Microtus arvalis) samples infected with two TULV strains each allowed for the de novo assembly of all four TULV genomes. Combining the novel sequences with all available TULV and PUUV genomes revealed very similar patterns of sequence diversity along the genomes, except for remarkably higher diversity in the non-coding region of the S-segment in PUUV. The genomic distribution of polymorphisms in the coding sequence was similar between the species, but differed between the segments with the highest sequence divergence of 0.274 for the M-segment, 0.265 for the S-segment, and 0.248 for the L-segment (overall 0.258). Phylogenetic analyses showed the clustering of genome sequences consistent with their geographic distribution within each species. Genome-wide data yielded extremely high node support values, despite the impact of strong mutational saturation that is expected for hantavirus sequences obtained over large spatial distances. We conclude that genome sequencing based on capture enrichment protocols provides an efficient means for ecological and evolutionary investigations of hantaviruses at an unprecedented completeness and depth.

Monkeypox is a viral zoonotic disease on the rise across endemic habitats. Despite the growing importance of monkeypox virus, our knowledge on its host spectrum and sylvatic maintenance is limited. Here, we describe the recent repeated emergence of monkeypox virus in a wild, human-habituated western chimpanzee (Pan troglodytes verus, hereafter chimpanzee) population from Taï National Park, Ivory Coast. Through daily monitoring, we show that further to causing its typical exanthematous syndrome, monkeypox can present itself as a severe respiratory disease without a diffuse rash. By analysing 949 non-invasively collected samples, we identify the circulation of at least two distinct monkeypox virus lineages and document the shedding of infectious particles in faeces and flies, suggesting that they could mediate indirect transmission. We also show that the carnivorous component of the Taï chimpanzees’ diet, mainly consisting of the sympatric monkeys they regularly hunt, did not change nor shift towards rodent consumption (the presumed reservoir) before the outbreaks, suggesting that the sudden emergence of monkeypox virus in this population is probably due to changes in the ecology of the virus itself. Using long-term mortality surveillance data from Taï National Park, we provide evidence of little to no prior viral activity over at least two decades. We conclude that great ape sentinel systems devoted to the longitudinal collection of behavioural and health data can help clarify the epidemiology and clinical presentation of zoonotic pathogens.

Abstract Baits targeting invertebrate ultraconserved elements (UCEs) are becoming more common for phylogenetic studies. Recent studies have shown that invertebrate UCEs typically encode proteins—and thus, are functionally different from more conserved vertebrate UCEs—and can resolve deep divergences (e.g., superorder to family ranks). However, whether invertebrate UCE baits have the power to robustly resolve relationships at shallower phylogenetic scales has been generally limited to investigations within the Coleoptera and Hymenoptera; thus, there are many invertebrate UCE baits that remain to be tested at shallower levels (i.e., tribes and congeners). Here, we assessed the ability of a recently designed Hemiptera UCE bait set to reconstruct more recent phylogenetic relationships in the largest leaf-footed bug subfamily, the Coreinae (Hemiptera: Coreidae), using a taxon-rich sample representing 21 of the 32 coreine tribes. Many well-supported, novel relationships were congruent in maximum likelihood and summary coalescent analyses. We also found evidence for the para- and polyphyly of several tribes and genera of Coreinae, as well as the subfamilies Coreinae and Meropachyinae. Our study, along with other recent UCE studies, provides evidence that UCEs can produce robust and novel phylogenetic hypotheses at various scales in invertebrates.

Longitudinal single-cell analysis of transcription and DNA methylation dynamics in cancer cell lines suggests a clonally stable epigenetic memory. Colon cancer cells show a spectrum of epithelial-to-mesenchymal identities that seems independent of genetic variation.