BackgroundThe de novo assembly of repeat-rich mammalian genomes using only high-throughput short read sequencing data typically results in highly fragmented genome assemblies that limit downstream applications. Here, we present an iterative approach to hybrid de novo genome assembly that incorporates datasets stemming from multiple genomic technologies and methods. We used this approach to improve the gray mouse lemur (Microcebus murinus) genome from early draft status to a near chromosome-scale assembly.MethodsWe used a combination of advanced genomic technologies to iteratively resolve conflicts and super-scaffold the M. murinus genome.ResultsWe improved the M. murinus genome assembly to a scaffold N50 of 93.32 Mb. Whole genome alignments between our primary super-scaffolds and 23 human chromosomes revealed patterns that are congruent with historical comparative cytogenetic data, thus demonstrating the accuracy of our de novo scaffolding approach and allowing assignment of scaffolds to M. murinus chromosomes. Moreover, we utilized our independent datasets to discover and characterize sequences associated with centromeres across the mouse lemur genome. Quality assessment of the final assembly found 96% of mouse lemur canonical transcripts nearly complete, comparable to other published high-quality reference genome assemblies.ConclusionsWe describe a new assembly of the gray mouse lemur (Microcebus murinus) genome with chromosome-scale scaffolds produced using a hybrid bioinformatic and sequencing approach. The approach is cost effective and produces superior results based on metrics of contiguity and completeness. Our results show that emerging genomic technologies can be used in combination to characterize centromeres of non-model species and to produce accurate de novo chromosome-scale genome assemblies of complex mammalian genomes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-017-0439-6) contains supplementary material, which is available to authorized users.

SARS-CoV-2 has spread rapidly across the world and is negatively impacting the global human population. COVID-19 patients display a wide variety of symptoms and clinical outcomes, including those attributed to genetic ancestry. Alu retrotransposons have played an important role in human evolution, and their variants influence host response to viral infection. Intronic Alus regulate gene expression through several mechanisms, including both genetic and epigenetic pathways. With respect to SARS-CoV-2, an intronic Alu within the ACE gene is hypothesized to be associated with COVID-19 susceptibility and morbidity. Here, we review specific Alu polymorphisms that are of particular interest when considering host response to SARS-CoV-2 infection, especially polymorphic Alu insertions in genes associated with immune response and coagulation/fibrinolysis cascade. We posit that additional research focused on Alu-related pathways could yield novel biomarkers capable of predicting clinical outcomes as well as patient-specific treatment strategies for COVID-19 and related infectious diseases.

Zoonotic diseases are a looming threat to global populations, and nearly 75% of emerging infectious diseases can spread among wildlife, domestic animals and humans. A 'One World, One Health' perspective offers us an ideal framework for understanding and potentially mitigating the spread of zoonoses, and the island of Madagascar serves as a natural laboratory for conducting these studies. Rapid habitat degradation and climate change on the island are contributing to more frequent contact among humans, livestock and wildlife, increasing the potential for pathogen spillover events. Given Madagascar's long geographical isolation, coupled with recent and repeated introduction of agricultural and invasive species, it is likely that a number of circulating pathogens remain uncharacterized in lemur populations. Thus, it is imperative that new approaches be implemented for de novo pathogen discovery. To this end, we used nontargeted deep sequencing of blood transcriptomes from two species of critically endangered wild lemurs (Indri indri and Propithecus diadema) to characterize blood-borne pathogens. Our results show several undescribed vector-borne parasites circulating within lemurs, some of which may cause disease in wildlife, livestock and humans. We anticipate that advanced methods for de novo identification of unknown pathogens will have broad utility for characterizing other complex disease transmission systems.

The golden lion tamarin (Leontopithecus rosalia) is an endangered Platyrrhine primate endemic to the Atlantic coastal forests of Brazil. Despite ongoing conservation efforts, genetic data on this species remains scarce. Complicating factors include limitations on sample collection and a lack of high-quality reference sequences. Here, we used nanopore adaptive sampling to resequence the L. rosalia mitogenome from feces, a sample which can be collected non-invasively. Adaptive sampling doubled the fraction of both host-derived and mitochondrial sequences compared to sequencing without enrichment. 258x coverage of the L. rosalia mitogenome was achieved in a single flow cell by targeting the unfinished genome of the distantly related emperor tamarin (Saguinus imperator) and the mitogenome of the closely related black lion tamarin (Leontopithecus chrysopygus). The L. rosalia mitogenome has a length of 16,597 bp, sharing 99.68% sequence identity with the L. chrysopygus mitogenome. A total of 38 SNPs between them were identified, with the majority being found in the non-coding D-loop region. DNA methylation and hydroxymethylation were directly detected using a neural network model applied to the raw signal from the MinION sequencer. In contrast to prior reports, DNA methylation was negligible in mitochondria in both CpG and non-CpG contexts. Surprisingly, a quarter of the 642 CpG sites exhibited DNA hydroxymethylation greater than 1% and 44 sites were above 5%, with concentration in the 3' side of several coding regions. Overall, we report a robust new mitogenome assembly for L. rosalia and direct detection of cytosine base modifications in all contexts.

Chronic wasting disease (CWD) is a disease affecting cervids and is caused by prions accumulating as pathogenic fibrils in lymphoid tissue and the central nervous system. Approaches for detecting CWD prions historically relied on antibody-based assays. However, recent advancements in protein amplification technology provided the foundation for a new class of CWD diagnostic tools. In particular, real-time quaking-induced conversion (RT-QuIC) has rapidly become a feasible option for CWD diagnosis. Despite its increased usage for CWD-focused research, there lacks a consensus regarding the interpretation of RT-QuIC data for diagnostic purposes. It is imperative then to identify a standardized and replicable method for determining CWD status from RT-QuIC data. Here, we assessed variables that could impact RT-QuIC results and explored the use of maxpoint ratios (maximumRFU/backgroundRFU) to improve the consistency of RT-QuIC analysis. We examined a variety of statistical analyses to retrospectively analyze CWD status based on RT-QuIC and ELISA results from 668 white-tailed deer lymph nodes. Our results revealed an MPR threshold of 2.0 for determining the rate of amyloid formation, and MPR analysis showed excellent agreement with independent ELISA results. These findings suggest that the use of MPR is a statistically viable option for normalizing between RT-QuIC experiments and defining CWD status.

Technological and computational advancements in the fields of genomics and bioinformatics are providing exciting new opportunities for pathogen discovery and genomic surveillance. In particular, single-molecule nucleotide sequence data originating from Oxford Nanopore Technologies (ONT) sequencing platforms can be bioinformatically leveraged, in real-time, for enhanced biosurveillance of a vast array of zoonoses. The recently released nanopore adaptive sampling (NAS) strategy facilitates immediate mapping of individual nucleotide molecules to a given reference as each molecule is being sequenced. User-defined thresholds then allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. Here, we show how NAS can be used to selectively sequence DNA of multiple bacterial tick-borne pathogens circulating in wild populations of the blacklegged tick vector, Ixodes scapularis.

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Nucleotide excision repair is the primary DNA repair mechanism that removes bulky DNA adducts such as UV-induced pyrimidine dimers. Correspondingly, genome-wide mapping of nucleotide excision repair with eXcision Repair sequencing (XR-seq), provides comprehensive pro ling of DNA damage repair. A number of XR-seq experiments at a variety of conditions for di erent damage types revealed heterogenous repair in the human genome. Although human repair pro les were extensively studied, how repair maps vary between primates is yet to be investigated. Here, we characterized the genome-wide UV-induced damage repair in gray mouse lemur,Microcebus murinus, in comparison to human. Mouse lemurs are strictly nocturnal, are the world's smallest living primates, and last shared a common ancestor with humans at least 60 million years ago. We derived broblast cell lines from mouse lemur, exposed them to UV irradiation. The following repair events were captured genome-wide through the XR-seq protocol. Mouse lemur repair pro les were analyzed in comparison to the equivalent human broblast datasets. We found that overall UV sensitivity, repair e ciency, and transcription-coupled repair levels di er between the two primates. Despite this, comparative analysis of human and mouse lemur broblasts revealed that genome-wide repair pro les of the homologous regions are highly correlated. This correlation is stronger for the highly expressed genes. With the inclusion of an additional XR-seq sample derived from another human cell line in the analysis, we found that broblasts of the two primates repair UV-induced DNA lesions in a more similar pattern than two distinct human cell lines do. Our results suggest that mouse lemurs and humans, and possibly primates in general, share a homologous repair mechanism as well as genomic variance distribution, albeit with their variable repair e ciency. This result also emphasizes the deep homologies of individual tissue types across the eukaryotic phylogeny.