Characterisation of Miscanthus genetic resources: a combined analysis of plastid and nuclear microsatellites, nrDNA sequences, flow cytometry and morphology.

Doctoral ThesisMiscanthus is a highly important forage and horticultural genus of perennial grasses (Poaceae) primarily native to South East Asia. Miscanthus is under intense global investigation as a biomass source for renewable energy production and several breeding initiatives are underway to develop new genotypes optimized for improved biomass and tolerance to a range of environmental stress conditions. A collection of 128 accessions belonging to the genus Miscanthus was established in Oak Park, Teagasc, Carlow, in 2008 and was investigated for morphological and molecular variation. Morphological traits were measured at the end of the second growing season and were compared with herbarium specimens of Miscanthus. Vegetative and inflorescence traits were scored and analysed using basic summary statistics, tests of normality and Principal Components Analysis (PCA). A large degree of morphological variation was recorded in the collections. The PCA of herbarium specimens was able to separate some species from others but there was also considerable overlap among species in the ordination, especially M. sacchariflorus, M. sinensis, M. condensatus and M. floridulus. These are known to be closely related and can interbreed. The PCA of the specimens from the Oak Park collection was less informative because of missing data due to lack of inflorescences (accessions did not flower). It was clear that morphology alone is often insufficient to distinguish taxa especially when inflorescence characters and ploidy information is lacking. The ploidy level of the accessions in the collection was evaluated through flow cytometry. The ploidy included di-, tri- and tetraploids. All individuals labelled as M. ×giganteus showed a triploid status, together with the newly bred M. sacchariflorus×M. sinensis hybrids. Most M. sinensis were diploids. Miscanthus sinensis Tea-62 was triploid and comparable to the value of the M. ×giganteus. A different situation was found for other non-diploid M. sinensis, in particular four M. sinensis ‘Goliath’ and the M. sinensis ‘Zebrinus’ Tea-33. In these the ratio measured by the flowcytometer was in between the values of the triploid M. giganteus and tetraploid M. sacchariflorus standards. The ‘Goliath-like’ hybrid is likely an autotriploid with three M. sinensis haploid sets, whereas M. ×giganteus is an allotriploid that is supposed to have two genomes from M. sinensis and one from M. sacchariflorus, which has a lower amount of DNA per haploid genome. DNA sequences of the internal transcribed spacer of the nrDNA were obtained for 76 genotypes in the collection and compared for polymorphism. The SNPs were particularly VI useful for differentiating M. sinensis, M. sacchariflorus and M. ×giganteus accessions and in combination with ploidy and morphology offer high potential for taxon identification. To gather more markers for population level diversity and differentiation studies, new microsatellite markers for both plastid and nuclear genomes were developed. For the development of plastid markers the chloroplast genome information of Saccharum officinarum was used. The nuclear SSRs (nSSRs) were developed from the sequences of 192 clones obtained from microsatellite enriched library. New primer pairs for the amplification of nineteen nuclear loci and six chloroplast loci were developed. Both chloroplast (cpSSR) and nSSR primers were used to characterise DNA variation, to help establish gene pools and to better understand hybridization and introgression. Huge genotypic variation was found within the genus, mostly in the species M. sinensis. The markers showed wide utility across a large number of Miscanthus species and also some closely related genera. The analysis of the cpSSRs showed a high number of different haplotypes but with a clear bias in allele composition between M. sinensis and the two species M. sacchariflorus and M. ×giganteus, thus confirming M. sacchariflorus as the maternal lineage of the hybrid M. xgiganteus. The nSSRs were found to be highly polymorphic across the collection and transferable to closely related genera such as Saccharum. The new markers were also used in UPGMA clustering and Bayesian structuring analysis to group individuals according to their similarity. Three major clusters of individuals were defined using the Bayesian STRUCTURE analysis with nuclear markers (nSSRs) and two with plastid markers (cpSSRs). In conclusion, the morphological, ploidy, sequence and microsatellite results highlighted the high level of diversity still unexplored in the genus and have clarified taxon identity of many accessions in the collection. A large set of new markers have been developed for the plant breeding and systematics community. The newly developed markers will be useful to further explore this diversity and to select useful traits for breeding of new and improved genotypes for biomass production.Teagasc Walsh Fellowship; National Development Plan Irelan

Targeted capture and sequencing of Orientia tsutsugamushi genomes from chiggers and humans

Scrub typhus is a febrile disease caused by Orientia tsutsugamushi, transmitted by larval stage Trombiculid mites (chiggers), whose primary hosts are small mammals. The phylogenomics of O. tsutsugamushi in chiggers, small mammals and humans remains poorly understood. To combat the limitations imposed by the low relative quantities of pathogen DNA in typical O. tsutsugamushi clinical and ecological samples, along with the technical, safety and cost limitations of cell culture, a novel probe-based target enrichment sequencing protocol was developed. The method was designed to capture variation among conserved genes and facilitate phylogenomic analysis at the scale of population samples. A whole-genome amplification step was incorporated to enhance the efficiency of sequencing by reducing duplication rates. This resulted in on-target capture rates of up to 93% for a diverse set of human, chigger, and rodent samples, with the greatest success rate in samples with real-time PCR Ct values below 35. Analysis of the best-performing samples revealed phylogeographic clustering at local, provincial and international scales. Applying the methodology to a comprehensive set of samples could yield a more complete understanding of the ecology, genomic evolution and population structure of O. tsutsugamushi and other similarly challenging organisms, with potential benefits in the development of diagnostic tests and vaccines

MinION Analysis and Reference Consortium: Phase 1 data release and analysis

The advent of a miniaturized DNA sequencing device with a high-throughput contextual sequencing capability embodies the next generation of large scale sequencing tools. The MinION™ Access Programme (MAP) was initiated by Oxford Nanopore Technologies™ in April 2014, giving public access to their USB-attached miniature sequencing device. The MinION Analysis and Reference Consortium (MARC) was formed by a subset of MAP participants, with the aim of evaluating and providing standard protocols and reference data to the community. Envisaged as a multi-phased project, this study provides the global community with the Phase 1 data from MARC, where the reproducibility of the performance of the MinION was evaluated at multiple sites. Five laboratories on two continents generated data using a control strain of Escherichia coli K-12, preparing and sequencing samples according to a revised ONT protocol. Here, we provide the details of the protocol used, along with a preliminary analysis of the characteristics of typical runs including the consistency, rate, volume and quality of data produced. Further analysis of the Phase 1 data presented here, and additional experiments in Phase 2 of E. coli from MARC are already underway to identify ways to improve and enhance MinION performance

Phylogenetic Analysis of Hepatitis C Virus Infections in a Large Belgian Cohort Using Next-Generation Sequencing of Full-Length Genomes

Funding Information: This research and Kasper Thorhauge Christensen was funded by grants from the Fonds Wetenschappelijk Onderzoek Vlaanderen (FWO) (G069214, G0B2317N and 1S38819N). Lize Cuypers acknowledges FWO travel grant for a research visit at the University of Oxford (V431117N). Core funding to the Wellcome Centre for Human Genetics was provided by the Wellcome Trust (award 203141/Z/16/Z). The authors acknowledge the STOP-HCV consortium that was funded by a grant from the Medical Research Council, United Kingdom (MR/K01532X/1). Publisher Copyright: © 2023 by the authors.The hepatitis C virus (HCV) epidemic in Western countries is primarily perpetuated by the sub-populations of men who have sex with men (MSM) and people who inject drugs (PWID). Understanding the dynamics of transmission in these communities is crucial for removing the remaining hurdles towards HCV elimination. We sequenced 269 annotated HCV plasma samples using probe enrichment and next-generation sequencing, obtaining 224 open reading frames of HCV (OR497849-OR498072). Maximum likelihood phylogenies were generated on the four most prevalent subtypes in this study (HCV1a, 1b, 3a, 4d) with a subsequent transmission cluster analysis. The highest rate of clustering was observed for HCV4d samples (13/17 (76.47%)). The second highest rate of clustering was observed in HCV1a samples (42/78 (53.85%)) with significant association with HIV-positive MSM. HCV1b and HCV3a had very low rates of clustering (2/83 (2.41%) and (0/29)). The spread of the prevalent subtype HCV1b appears to have been largely curtailed, and we demonstrate the onwards transmission of HCV1a and HCV4d in the HIV-positive MSM population across municipal borders. More systematic data collection and sequencing is needed to allow a better understanding of the HCV transmission among the community of PWID and overcome the remaining barriers for HCV elimination in Belgium.publishersversionpublishe

Castanet: a pipeline for rapid analysis of targeted multi-pathogen genomic data

Motivation: Target enrichment strategies generate genomic data from multiple pathogens in a single process, greatly improving sensitivity over metagenomic sequencing and enabling cost-effective, high-throughput surveillance and clinical applications. However, uptake by research and clinical laboratories is constrained by an absence of computational tools that are specifically designed for the analysis of multi-pathogen enrichment sequence data. Here we present an analysis pipeline, Castanet, for use with multi-pathogen enrichment sequencing data. Castanet is designed to work with short-read data produced by existing targeted enrichment strategies, but can be readily deployed on any BAM file generated by another methodology. Also included are an optional graphical interface and installer script. Results: In addition to genome reconstruction, Castanet reports method-specific metrics that enable quantification of capture efficiency, estimation of pathogen load, differentiation of low-level positives from contamination, and assessment of sequencing quality. Castanet can be used as a traditional end-to-end pipeline for consensus generation, but its strength lies in the ability to process a flexible, pre-defined set of pathogens of interest directly from multi-pathogen enrichment experiments. In our tests, Castanet consensus sequences were accurate reconstructions of reference sequences, including in instances where multiple strains of the same pathogen were present. Castanet performs effectively on standard computers and can process the entire output of a 96-sample enrichment sequencing run (50M reads) using a single batch process command, in

Targeted metagenomics reveals association between severity and pathogen co-detection in infants with respiratory syncytial virus

Respiratory syncytial virus (RSV) is the leading cause of hospitalisation for respiratory infection in young children. RSV disease severity is known to be age-dependent and highest in young infants, but other correlates of severity, particularly the presence of additional respiratory pathogens, are less well understood. In this study, nasopharyngeal swabs were collected from two cohorts of RSV-positive infants 100 pathogens, including all common respiratory viruses and bacteria, from samples collected from 433 infants, that burden of additional viruses is common (111/433, 26%) but only modestly correlates with RSV disease severity. In contrast, there is strong evidence in both cohorts and across age groups that presence of Haemophilus bacteria (194/433, 45%) is associated with higher severity, including much higher rates of hospitalisation (odds ratio 4.25, 95% CI 2.03–9.31). There is no evidence for association between higher severity and other detected bacteria, and no difference in severity between RSV genotypes. Our findings reveal the genomic diversity of additional pathogens during RSV infection in infants, and provide an evidence base for future causal investigations of the impact of co-infection on RSV disease severity

The mutational spectrum of SARS-CoV-2 genomic and antigenomic RNA

The raw material for viral evolution is provided by intra-host mutations occurring during replication, transcription or post-transcription. Replication and transcription of Coronaviridae proceed through the synthesis of negative-sense ‘antigenomes’ acting as templates for positive-sense genomic and subgenomic RNA. Hence, mutations in the genomes of SARS-CoV-2 and other coronaviruses can occur during (and after) the synthesis of either negative-sense or positive-sense RNA, with potentially distinct patterns and consequences. We explored for the first time the mutational spectrum of SARS-CoV-2 (sub)genomic and anti(sub)genomic RNA. We use a high-quality deep sequencing dataset produced using a quantitative strand-aware sequencing method, controlled for artefacts and sequencing errors, and scrutinized for accurate detection of within-host diversity. The nucleotide differences between negative- and positive-sense strand consensus vary between patients and do not show dependence on age or sex. Similarities and differences in mutational patterns between within-host minor variants on the two RNA strands suggested strand-specific mutations or editing by host deaminases and oxidative damage. We observe generally neutral and slight negative selection on the negative strand, contrasting with purifying selection in ORF1a, ORF1b and S genes of the positive strand of the genome

Evidence of tenofovir resistance in chronic hepatitis B virus (HBV) infection: An observational case series of South African adults

Introduction: Tenofovir disoproxil fumarate (TDF) is widely recommended for treatment of chronic hepatitis B virus (HBV) infection because it is safe, affordable and has a high genetic barrier to resistance. TDF resistance associated mutations (RAMs) have been reported, but data are limited, particularly for Africa. We set out to identify potential RAMs in individuals with detectable HBV viraemia on TDF treatment. Methods: We recruited adults with chronic HBV infection from Cape Town, South Africa, identifying individuals with a TDF resistance phenotype, defined as persistent HBV vireamia despite >12 months of TDF treatment. We sequenced HBV DNA using MiSeq Illumina with whole genome target enrichment, and sought potential TDF RAMs, based on a pre-defined list of polymorphisms. Results: Among 66 individuals with chronic HBV (genotypes A and D), three met our clinical definition for TDF resistance, of whom two were coinfected with HIV. In one participant, the consensus HBV sequence contained nine polymorphisms that have been described in association with TDF resistance. Significant treatment non-adherence in this individual was unlikely, as HIV RNA was suppressed. TDF RAMs were also present in HBV sequences from the other two participants, but other factors including treatment non-adherence may also have had a role in failure of HBV DNA suppression in these cases. Discussion: Our findings add to the evidence that RAMs in HBV reverse transcriptase may underpin a TDF resistant phenotype. This is the first time these RAMs have been reported from Africa in association with clinical evidence of TDF resistance

A highly virulent variant of HIV-1 circulating in the Netherlands

We discovered a highly virulent variant of subtype-B HIV-1 in the Netherlands. One hundred nine individuals with this variant had a 0.54 to 0.74 log(10) increase (i.e., a similar to 3.5-fold to 5.5-fold increase) in viral load compared with, and exhibited CD4 cell decline twice as fast as, 6604 individuals with other subtype-B strains. Without treatment, advanced HIV-CD4 cell counts below 350 cells per cubic millimeter, with long-term clinical consequences-is expected to be reached, on average, 9 months after diagnosis for individuals in their thirties with this variant. Age, sex, suspected mode of transmission, and place of birth for the aforementioned 109 individuals were typical for HIV-positive people in the Netherlands, which suggests that the increased virulence is attributable to the viral strain. Genetic sequence analysis suggests that this variant arose in the 1990s from de novo mutation, not recombination. with increased transmissibility and an unfamiliar molecular mechanism of virulence