Detection of Mpox Virus Using Microbial Cell-Free DNA: The Potential of Pathogen-Agnostic Sequencing for Rapid Identification of Emerging Pathogens.

BACKGROUND: The 2022 global outbreak of Monkeypox virus (MPXV) highlighted challenges with polymerase chain reaction detection as divergent strains emerged and atypical presentations limited the applicability of swab sampling. Recommended testing in the United States requires a swab of lesions, which arise late in infection and may be unrecognized. We present MPXV detections using plasma microbial cell-free DNA (mcfDNA) sequencing. METHODS: Fifteen plasma samples from 12 case-patients were characterized through mcfDNA sequencing. Assay performance was confirmed through in silico inclusivity and exclusivity assessments. MPXV isolates were genotyped using mcfDNA, and phylodynamic information was imputed using publicly available sequences. RESULTS: MPXV mcfDNA was detected in 12 case-patients. Mpox was not suspected in 5, with 1 having documented resolution of mpox >6 months previously. Six had moderate to severe mpox, supported by high MPXV mcfDNA concentrations; 4 died. In 7 case-patients, mcfDNA sequencing detected coinfections. Genotyping by mcfDNA sequencing identified 22 MPXV mutations at 10 genomic loci in 9 case-patients. Consistent with variation observed in the 2022 outbreak, 21 of 22 variants were G > A/C > T. Phylogenetic analyses imputed isolates to sublineages arising at different time points and from different geographic locations. CONCLUSIONS: We demonstrate the potential of plasma mcfDNA sequencing to detect, quantify, and, for acute infections with high sequencing coverage, subtype MPXV using a single noninvasive test. Sequencing plasma mcfDNA may augment existing mpox testing in vulnerable patient populations or in patients with atypical symptoms or unrecognized mpox. Strain type information may supplement disease surveillance and facilitate tracking emerging pathogens.

A chromosome conformation capture ordered sequence of the barley genome.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

The genome of cowpea (Vigna unguiculata [L.] Walp.).

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub-Saharan Africa, that is resilient to hot and drought-prone environments. An assembly of the single-haplotype inbred genome of cowpea IT97K-499-35 was developed by exploiting the synergies between single-molecule real-time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination-poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high-recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS-LRR and the SAUR-like auxin superfamilies compared with other warm-season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.

Higher classification sensitivity of short metagenomic reads with CLARK-S.

The growing number of metagenomic studies in medicine and environmental sciences is creating increasing demands on the computational infrastructure designed to analyze these very large datasets. Often, the construction of ultra-fast and precise taxonomic classifiers can compromise on their sensitivity (i.e. the number of reads correctly classified). Here we introduce CLARK-S, a new software tool that can classify short reads with high precision, high sensitivity and high speed. AVAILABILITY AND IMPLEMENTATION: CLARK-S is freely available at http://clark.cs.ucr.edu/ CONTACT: stelo@cs.ucr.eduSupplementary information: Supplementary data are available at Bioinformatics online.

BRAT-nova: fast and accurate mapping of bisulfite-treated reads.

UNLABELLED: In response to increasing amounts of sequencing data, faster and faster aligners need to become available. Here, we introduce BRAT-nova, a completely rewritten and improved implementation of the mapping tool BRAT-BW for bisulfite-treated reads (BS-Seq). BRAT-nova is very fast and accurate. On the human genome, BRAT-nova is 2-7 times faster than state-of-the-art aligners, while maintaining the same percentage of uniquely mapped reads and space usage. On synthetic reads, BRAT-nova is 2-8 times faster than state-of-the-art aligners while maintaining similar mapping accuracy, methylation call accuracy, methylation level accuracy and space efficiency. AVAILABILITY AND IMPLEMENTATION: The software is available in the public domain at http://compbio.cs.ucr.edu/brat/ CONTACT: elenah@cs.ucr.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

rasbhari: Optimizing Spaced Seeds for Database Searching, Read Mapping and Alignment-Free Sequence Comparison.

Many algorithms for sequence analysis rely on word matching or word statistics. Often, these approaches can be improved if binary patterns representing match and don't-care positions are used as a filter, such that only those positions of words are considered that correspond to the match positions of the patterns. The performance of these approaches, however, depends on the underlying patterns. Herein, we show that the overlap complexity of a pattern set that was introduced by Ilie and Ilie is closely related to the variance of the number of matches between two evolutionarily related sequences with respect to this pattern set. We propose a modified hill-climbing algorithm to optimize pattern sets for database searching, read mapping and alignment-free sequence comparison of nucleic-acid sequences; our implementation of this algorithm is called rasbhari. Depending on the application at hand, rasbhari can either minimize the overlap complexity of pattern sets, maximize their sensitivity in database searching or minimize the variance of the number of pattern-based matches in alignment-free sequence comparison. We show that, for database searching, rasbhari generates pattern sets with slightly higher sensitivity than existing approaches. In our Spaced Words approach to alignment-free sequence comparison, pattern sets calculated with rasbhari led to more accurate estimates of phylogenetic distances than the randomly generated pattern sets that we previously used. Finally, we used rasbhari to generate patterns for short read classification with CLARK-S. Here too, the sensitivity of the results could be improved, compared to the default patterns of the program. We integrated rasbhari into Spaced Words; the source code of rasbhari is freely available at http://rasbhari.gobics.de/.

Sequencing of 15 622 gene-bearing BACs clarifies the gene-dense regions of the barley genome.

Barley (Hordeum vulgare L.) possesses a large and highly repetitive genome of 5.1 Gb that has hindered the development of a complete sequence. In 2012, the International Barley Sequencing Consortium released a resource integrating whole-genome shotgun sequences with a physical and genetic framework. However, because only 6278 bacterial artificial chromosome (BACs) in the physical map were sequenced, fine structure was limited. To gain access to the gene-containing portion of the barley genome at high resolution, we identified and sequenced 15 622 BACs representing the minimal tiling path of 72 052 physical-mapped gene-bearing BACs. This generated ~1.7 Gb of genomic sequence containing an estimated 2/3 of all Morex barley genes. Exploration of these sequenced BACs revealed that although distal ends of chromosomes contain most of the gene-enriched BACs and are characterized by high recombination rates, there are also gene-dense regions with suppressed recombination. We made use of published map-anchored sequence data from Aegilops tauschii to develop a synteny viewer between barley and the ancestor of the wheat D-genome. Except for some notable inversions, there is a high level of collinearity between the two species. The software HarvEST:Barley provides facile access to BAC sequences and their annotations, along with the barley-Ae. tauschii synteny viewer. These BAC sequences constitute a resource to improve the efficiency of marker development, map-based cloning, and comparative genomics in barley and related crops. Additional knowledge about regions of the barley genome that are gene-dense but low recombination is particularly relevant.

CLARK: fast and accurate classification of metagenomic and genomic sequences using discriminative k-mers.

BACKGROUND: The problem of supervised DNA sequence classification arises in several fields of computational molecular biology. Although this problem has been extensively studied, it is still computationally challenging due to size of the datasets that modern sequencing technologies can produce. RESULTS: We introduce CLARK a novel approach to classify metagenomic reads at the species or genus level with high accuracy and high speed. Extensive experimental results on various metagenomic samples show that the classification accuracy of CLARK is better or comparable to the best state-of-the-art tools and it is significantly faster than any of its competitors. In its fastest single-threaded mode CLARK classifies, with high accuracy, about 32 million metagenomic short reads per minute. CLARK can also classify BAC clones or transcripts to chromosome arms and centromeric regions. CONCLUSIONS: CLARK is a versatile, fast and accurate sequence classification method, especially useful for metagenomics and genomics applications. It is freely available at http://clark.cs.ucr.edu/ .

Ozone Disinfection for Elimination of Bacteria and Degradation of SARS-CoV2 RNA for Medical Environments.

Pathogenic bacteria and viruses in medical environments can lead to treatment complications and hospital-acquired infections. Current disinfection protocols do not address hard-to-access areas or may be beyond line-of-sight treatment, such as with ultraviolet radiation. The COVID-19 pandemic further underscores the demand for reliable and effective disinfection methods to sterilize a wide array of surfaces and to keep up with the supply of personal protective equipment (PPE). We tested the efficacy of Sani Sport ozone devices to treat hospital equipment and surfaces for killing Escherichia coli, Enterococcus faecalis, Bacillus subtilis, and Deinococcus radiodurans by assessing Colony Forming Units (CFUs) after 30 min, 1 h, and 2 h of ozone treatment. Further gene expression analysis was conducted on live E. coli K12 immediately post treatment to understand the oxidative damage stress response transcriptome profile. Ozone treatment was also used to degrade synthetic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA as assessed by qPCR CT values. We observed significant and rapid killing of medically relevant and environmental bacteria across four surfaces (blankets, catheter, remotes, and syringes) within 30 min, and up to a 99% reduction in viable bacteria at the end of 2 h treatment cycles. RNA-seq analysis of E. coli K12 revealed 447 differentially expressed genes in response to ozone treatment and an enrichment for oxidative stress response and related pathways. RNA degradation of synthetic SARS-CoV-2 RNA was seen an hour into ozone treatment as compared to non-treated controls, and a non-replicative form of the virus was shown to have significant RNA degradation at 30 min. These results show the strong promise of ozone treatment of surfaces for reducing the risk of hospital-acquired infections and as a method for degradation of SARS-CoV-2 RNA.

Characterization of Spacesuit Associated Microbial Communities and Their Implications for NASA Missions.

BACKGROUND: Crewed National Aeronautics and Space Administration (NASA) missions to other solar system bodies are currently being planned. One high-profile scientific focus during such expeditions would be life detection, specifically the discovery of past or present microbial life, if they exist. However, both humans and associated objects typically carry a high microbial burden. Thus, it is essential to distinguish between microbes brought with the expedition and those present on the exploring planets. Modern spacesuits are unique, customized spacecraft which provide protection, mobility and life support to crew during spacewalks, yet they vent, and the mobility of microbes through spacesuits has not been studied. RESULTS: To evaluate the microbial colonization of spacesuits, NASA used an Extravehicular Activity swab kit to examine viable microbial populations of 48 samples from spacesuits using both traditional microbiological methods and molecular sequencing methods. The cultivable microbial population ranged from below the detection limit to 9 × 102 colony forming units per 25 cm2 of sample and also significantly varied by the location. The cultivable microbial diversity was dominated by members of Bacillus, Arthrobacter, and Ascomycota. However, 16S rRNA-based viable bacterial burden ranged from 105 to 106 copies per 25 cm2 of sample. Shotgun metagenome sequencing revealed the presence of a diverse microbial population on the spacesuit surfaces, including Curtobacterium and Methylobacterium from across all sets of spacesuits in high abundance. Among bacterial species identified, higher abundance of Cutibacterium acnes, Methylobacterium oryzae, and M. phyllosphaerae reads were documented. CONCLUSION: The results of this study provide evidence that identical microbial strains may live on the wrist joint, inner gauntlet, and outer gauntlet of spacesuits. This raises the possibility, but does not confirm that microbial contaminants on the outside of the suits could contaminate planetary science operations unless additional measures are taken. Overall, these data provide the first estimate of microbial distribution associated with spacesuit surfaces, which will help future mission planners develop effective planetary protection strategies.

Correction to: Comprehensive benchmarking and ensemble approaches for metagenomic classifiers.

Following publication of the original article [1], the authors would like to highlight the following two corrections.

Metagenomic characterization of ambulances across the USA.

BACKGROUND: Microbial communities in our built environments have great influence on human health and disease. A variety of built environments have been characterized using a metagenomics-based approach, including some healthcare settings. However, there has been no study to date that has used this approach in pre-hospital settings, such as ambulances, an important first point-of-contact between patients and hospitals. RESULTS: We sequenced 398 samples from 137 ambulances across the USA using shotgun sequencing. We analyzed these data to explore the microbial ecology of ambulances including characterizing microbial community composition, nosocomial pathogens, patterns of diversity, presence of functional pathways and antimicrobial resistance, and potential spatial and environmental factors that may contribute to community composition. We found that the top 10 most abundant species are either common built environment microbes, microbes associated with the human microbiome (e.g., skin), or are species associated with nosocomial infections. We also found widespread evidence of antimicrobial resistance markers (hits ~ 90% samples). We identified six factors that may influence the microbial ecology of ambulances including ambulance surfaces, geographical-related factors (including region, longitude, and latitude), and weather-related factors (including temperature and precipitation). CONCLUSIONS: While the vast majority of microbial species classified were beneficial, we also found widespread evidence of species associated with nosocomial infections and antimicrobial resistance markers. This study indicates that metagenomics may be useful to characterize the microbial ecology of pre-hospital ambulance settings and that more rigorous testing and cleaning of ambulances may be warranted.

Comprehensive benchmarking and ensemble approaches for metagenomic classifiers.

BACKGROUND: One of the main challenges in metagenomics is the identification of microorganisms in clinical and environmental samples. While an extensive and heterogeneous set of computational tools is available to classify microorganisms using whole-genome shotgun sequencing data, comprehensive comparisons of these methods are limited. RESULTS: In this study, we use the largest-to-date set of laboratory-generated and simulated controls across 846 species to evaluate the performance of 11 metagenomic classifiers. Tools were characterized on the basis of their ability to identify taxa at the genus, species, and strain levels, quantify relative abundances of taxa, and classify individual reads to the species level. Strikingly, the number of species identified by the 11 tools can differ by over three orders of magnitude on the same datasets. Various strategies can ameliorate taxonomic misclassification, including abundance filtering, ensemble approaches, and tool intersection. Nevertheless, these strategies were often insufficient to completely eliminate false positives from environmental samples, which are especially important where they concern medically relevant species. Overall, pairing tools with different classification strategies (k-mer, alignment, marker) can combine their respective advantages. CONCLUSIONS: This study provides positive and negative controls, titrated standards, and a guide for selecting tools for metagenomic analyses by comparing ranges of precision, accuracy, and recall. We show that proper experimental design and analysis parameters can reduce false positives, provide greater resolution of species in complex metagenomic samples, and improve the interpretation of results.

Construction of a map-based reference genome sequence for barley, Hordeum vulgare L.

Barley (Hordeum vulgare L.) is a cereal grass mainly used as animal fodder and raw material for the malting industry. The map-based reference genome sequence of barley cv. 'Morex' was constructed by the International Barley Genome Sequencing Consortium (IBSC) using hierarchical shotgun sequencing. Here, we report the experimental and computational procedures to (i) sequence and assemble more than 80,000 bacterial artificial chromosome (BAC) clones along the minimum tiling path of a genome-wide physical map, (ii) find and validate overlaps between adjacent BACs, (iii) construct 4,265 non-redundant sequence scaffolds representing clusters of overlapping BACs, and (iv) order and orient these BAC clusters along the seven barley chromosomes using positional information provided by dense genetic maps, an optical map and chromosome conformation capture sequencing (Hi-C). Integrative access to these sequence and mapping resources is provided by the barley genome explorer (BARLEX).