Whole Genome Sequencing Research Articles

Decoding the Root Lignification Mechanism of Angelica sinensis through Genome-wide Methylation Analysis.

Angelica sinensis is a traditional Chinese herbal medicine with significant economic and medicinal value. However, early bolting and flowering can occur during the second year of the vegetative growth period, rendering the roots unviable for medicinal use and resulting in substantial economic losses. Consequently, the growing interest in studying the molecular mechanisms underlying early bolting or increased root lignification in A. sinensis. Here, we conducted whole-genome bisulfite sequencing and observed an increase in whole-genome DNA methylation levels on chromosomes after bolting in A. sinensis. Comparative analysis revealed methylation patterns in the upstream, gene body, and downstream regions in the context of CG, CHG, and CHH, suggesting a possible association between CHH-type methylation of promoters and phenylpropanoid biosynthesis. Furthermore, joint analysis of transcriptomic and methylomics data revealed a positive correlation between DNA methylation and gene expression. We identified the hyperDMR gene in the CHH background within the promoter region; this gene is also a key gene (AsCOMT1), exhibiting dual catalytic activity and facilitating the synthesis of both ferulic acid and lignin. Enzyme kinetic analysis demonstrated that AsCOMT1 preferentially catalyzes the synthesis of lignin monomer precursors. These findings highlight the important regulatory role of DNA methylation in bolting and the synthesis of secondary metabolites in A. sinensis, providing valuable insights into the underlying molecular mechanisms. Therefore, as DNA methylation plays an important regulatory role in A. sinensis bolting and secondary metabolite synthesis, it has potential significance in the analysis of the underlying molecular mechanism.

438 DNA methylation patterns and transcriptional regulation during pig fetal skeletal muscle development

Abstract Fetal development is controlled by a complex cascade of highly regulated and coordinated gene expression patterns. Epigenetic mechanisms have important roles in regulating development and differentiation. Among such mechanisms, DNA methylation exhibits context-specific associations with gene expression and has been shown to be highly dynamic during developmental processes. We performed whole-genome bisulfite sequencing (WGBS) to assess DNA methylation in pig longissimus dorsi muscle at 41- and 70-d gestation (dg), as well as RNA- and small RNA-sequencing to identify coordinated changes in methylation and expression between myogenic stages. We identified 45,739 differentially methylated regions (DMRs) between stages, and the majority (n = 34,232) were hypomethylated at 70 vs. 41 dg. Developmental DMRs exhibited feature-specific enrichment in gene regulatory regions, as well as in regions proximal to micro-RNAs (miRNAs) that have known roles in myogenesis. Integration of methylation and transcriptomic data revealed strong associations between differential gene methylation and transcript abundance. We surveyed myogenic regulatory factor (MRF) genes to determine if differential methylation was present in expected genomic regions. Within the MYF5 and MYF6 locus, MYF5 was significantly promoter-hypermethylated at 70 dg, whereas MYF6 was significantly hypomethylated upstream of its transcription start site. MYF5 is the earliest MRF to be expressed and primarily functions in myoblast proliferation and determination, while MYF6 functions in muscle cell differentiation. Thus, these patterns were consistent with expected downregulation of MYF5 and upregulation of MYF6 as muscle development progresses and demonstrate that differential methylation is evident at myogenic transcription factors. Differential miRNA methylation was significantly negatively correlated with abundance, and dynamic expression of assayed miRNAs persisted postnatally. Motif analysis revealed significant enrichment of myogenic regulatory factor motifs among hypomethylated regions, suggesting that DNA hypomethylation may function to increase accessibility of muscle-specific transcription factors. We also show that developmental DMRs are enriched for GWAS SNPs associated with muscle physiology and meat quality traits, demonstrating the potential for epigenetic processes to influence phenotypic diversity. Our results enhance understanding of DNA methylation dynamics in pig fetal skeletal muscle and reveal putative cis-regulatory elements governed by epigenetic processes during porcine myogenesis.

Benchmarking bulk and single-cell variant-calling approaches on Chromium scRNA-seq and scATAC-seq libraries.

Single-cell sequencing methodologies such as scRNA-seq and scATAC-seq have become widespread and effective tools to interrogate tissue composition. Increasingly, variant callers are being applied to these methodologies to resolve the genetic heterogeneity of a sample, especially in the case of detecting the clonal architecture of a tumor. Typically, traditional bulk DNA variant callers are applied to the pooled reads of a single-cell library to detect candidate mutations. Recently, multiple studies have applied such callers on reads from individual cells, with some citing the ability to detect rare variants with higher sensitivity. Many studies apply these two approaches to the Chromium (10x Genomics) scRNA-seq and scATAC-seq methodologies. However, Chromium-based libraries may offer additional challenges to variant calling compared with existing single-cell methodologies, raising questions regarding the validity of variants obtained from such a workflow. To determine the merits and challenges of various variant-calling approaches on Chromium scRNA-seq and scATAC-seq libraries, we use sample libraries with matched bulk whole-genome sequencing to evaluate the performance of callers. We review caller performance, finding that bulk callers applied on pooled reads significantly outperform individual-cell approaches. We also evaluate variants unique to scRNA-seq and scATAC-seq methodologies, finding patterns of noise but also potential capture of RNA-editing events. Finally, we review the notion that variant calling at the single-cell level can detect rare somatic variants, providing empirical results that suggest resolving such variants is infeasible in single-cell Chromium libraries.

Genome-wide association mapping reveals novel genes and genomic regions controlling root-lesion nematode resistance in chickpea mini core collection.

Root-lesion nematodes (RLN) pose a significant threat to chickpea (Cicer arietinum L.) by damaging the root system and causing up to 25% economic losses due to reduced yield. Worldwide commercially grown chickpea varieties lack significant genetic resistance to RLN, necessitating the identification of genetic variants contributing to natural resistance. This study identifies genomic loci responsible for resistance to the RLN, Pratylenchus thornei Sher & Allen, in chickpea by utilizing high-quality single nucleotide polymorphisms from whole-genome sequencing data of 202 chickpea accessions. Phenotypic evaluations of the genetically diverse set of chickpea accessions in India and Australia revealed a wide range of responses from resistant to susceptible. Genome-wide association studies (GWAS) employing Fixed and Random Model Circulating Probability Unification (FarmCPU) and Bayesian-Information and Linkage-Disequilibrium Iteratively Nested Keyway (BLINK) models identified 44 marker-trait associations distributed across all chromosomes except Ca1. Crucially, genomic regions on Ca2 and Ca5 consistently display significant associations across locations. Of 25 candidate genes identified, five genes were putatively involved in RLN resistance response (glucose-6-phosphate dehydrogenase, heat shock proteins, MYB-like DNA-binding protein, zinc finger FYVE protein and pathogenesis-related thaumatin-like protein). One notably identified gene (Ca_10016) presents four haplotypes, where haplotypes 1-3 confer moderate susceptibility, and haplotype 4 contributes to high susceptibility to RLN. This information provides potential targets for marker development to enhance breeding for RLN resistance in chickpea. Additionally, five potential resistant genotypes (ICC3512, ICC8855, ICC5337, ICC8950, and ICC6537) to P. thornei were identified based on their performance at a specific location. The study's significance lies in its comprehensive approach, integrating multiple-location phenotypic evaluations, advanced GWAS models, and functional genomics to unravel the genetic basis of P. thornei resistance. The identified genomic regions, candidate genes, and haplotypes offer valuable insights for breeding strategies, paving the way for developing chickpea varieties resilient to P. thornei attack.

205 An autosomal recessive mutation in PYGM causes myophosphorylase deficiency in composite cattle

Abstract Between 2020 and 2022, eight calves (6 heifers, 2 bulls) in a Nebraska herd (composite Simmental, Red Angus, Gelbvieh) experienced exercise intolerance during extended physical activity. These calves exhibited an inability to keep pace with the rest of the herd, with some eventually succumbing to physical collapse from which they did not recover. Increased creatine kinase, an indication of muscle degeneration, was confirmed in biopsy and necropsy samples. Available sire pedigrees revealed a common paternal ancestor within 2 to 4 generations in all the affected calves. Dam pedigrees were unavailable; however, the herd retained replacement females that occasionally shared common ancestors with breeding bulls. Thus, a de novo autosomal recessive mutation was hypothesized as the cause of exercise intolerance in these calves. Genomic data (100K SNP) from 6 affected calves and 715 herd mates were initially used for a whole genome-wide association study, pinpointing a significant region on chromosome 29. Whole-genome sequencing of 2 affected calves combined with 145 other sequenced cattle led to the identification of a nonsense mutation in the PYGM gene, also located on chromosome 29. The mutation, confirmed to be present in the skeletal muscle transcriptome, was predicted to produce a premature stop codon. The protein product of PYGM, myophosphorylase, has a crucial role in metabolizing glycogen stored within skeletal muscle to generate energy for muscle cells. As a result, the recessive genotype led to greater (P < 0.05) glycogen concentrations in affected calves (n = 2) compared with heterozygous animals (n = 3) and wild-type controls (n = 2). Immunohistochemistry and label-free quantitative proteomics analysis confirmed the absence of the PYGM protein product in skeletal muscle. Myophosphorylase is needed for efficient glycogen utilization, which is crucial for producing high-quality beef. In the 2 affected calves harvested, increased skeletal muscle glycogen persisted postmortem, resulting in increased pH and dark-cutting beef, which is associated with a negative consumer perception and economic losses to the industry. In addition to identifying the cause of exercise intolerance and welfare concerns in these cattle, this study is among the first to identify a specific genetic mutation associated with dark-cutting beef. Carriers of the mutation did not display any discernible differences in meat quality or measures of animal well-being. While cattle heterozygous for the mutation may not immediately impact the beef industry, identifying carriers is paramount for implementing proactive selection and breeding strategies to prevent the production of affected calves.

Genomic profiling of cell-free DNA from dogs with benign and malignant tumors

ObjectiveCancer is currently the most common cause of death in adult dogs. Like humans, dogs have a one-third chance of developing cancer in their lifetime. We used shallow whole-genome sequencing (sWGS) to analyze blood cell-free DNA (cfDNA) from four tumor-bearing dogs (one with benign and three with malignant tumors) and 38 healthy dogs.ResultsSimilar to the results observed in the healthy dogs, no copy number aberration (CNA) was detected in the dog with benign lipomas, and the distribution of cfDNA fragment size (FS) closely resembled that of the healthy dogs. However, among the three dogs diagnosed with malignant tumors, two dogs exhibited varying degrees and quantities of CNAs. Compared to the distribution of FS in the healthy dogs, the cancer dogs exhibited a noticeable shift towards shorter lengths. These findings indicated that CNA and FS profiles derived from sWGS data can be used for non-invasive cancer detection in dogs.

High-speed whole-genome sequencing of a Whippet: Rapid chromosome-level assembly and annotation of an extremely fast dog’s genome

High-speed whole-genome sequencing of a Whippet: Rapid chromosome-level assembly and annotation of an extremely fast dog’s genome

Long-read DNA and cDNA sequencing identify cancer-predisposing deep intronic variation in tumor-suppressor genes.

The vast majority of deeply intronic genomic variants are benign, but some extremely rare or private deep intronic variants lead to exonification of intronic sequence with abnormal transcriptional consequences. Damaging variants of this class are likely underreported as causes of disease for several reasons: Most clinical DNA and RNA testing does not include full intronic sequences; many of these variants lie in complex repetitive regions that cannot be aligned from short-read whole-genome sequence; and, until recently, consequences of deep intronic variants were not accurately predicted by in silico tools. We evaluated the frequency and consequences of rare deep intronic variants for families severely affected with breast, ovarian, pancreatic, and/or metastatic prostate cancer, but with no causal variant identified by any previous genomic or cDNA-based approach. For 10 tumor-suppressor genes, we used multiplexed adaptive sampling long-read DNA sequencing and cDNA sequencing, based on patient-derived DNA and RNA, to systematically evaluate deep intronic variation. We identified all variants across the full genomic loci of targeted genes, applied the in silico tools SpliceAI and Pangolin to predict variants of functional consequence, and then carried out long-read cDNA sequencing to identify aberrant transcripts. For eight of the 120 (6%) previously unsolved families, rare deep intronic variants in BRCA1, PALB2, and ATM create intronic pseudoexons that are spliced into transcripts, leading to premature truncations. These results suggest that long-read DNA and cDNA sequencing can be integrated into variant discovery, with strategies for accurately characterizing pathogenic variants.

168 American mink Genome Sequencing Project

Abstract The long-term goal of American Mink (Neogale vison) Genome Sequencing Project is to develop novel genomics tools to study the genome biology and evolution of mink. This research agenda is organized around three major goals: 1) development of first chromosome-level genome assembly with high quality for American mink to help understanding the biology and evolution of the order Carnivora, 2) designing the first mink single nucleotide polymorphism (SNP) genotyping array for genomic discovery in mink, and 3) using genomics to understand the genetic architecture of economically important traits in mink. An initial draft of the genome assembly is generated using 2,884,047 PacBio long reads. Integration of Hi-C data into the initial draft led to an assembly with 183 scaffolds and scaffold N50 of 220 Mb. This gap-free genome assembly of American mink (ASM_NN_V1) has a length of 2.68 Gb in which about 98.6% of the whole genome is covered by 15 chromosomes. Significant improvements are observed in genome contiguity, the number of scaffolds, and annotation compared with the first draft of mink genome assembly. A custom genotyping array is developed using the developed mink reference genome and DNA panel composed of 100 whole-genome sequences data of American mink from five color-types. This Affymetrix Mink 70K SNP array created 70,000 SNPs, which is sufficient for conducting genomic studies in the mink research program and beyond. This project would facilitate the discovery of genomic regions, markers and genetic networks underlying economically important traits in American mink, which will contribute novel insights into the genetic architecture of key traits and is a critical step to implement genomic selection in mink breeding programs. The results of this research have the potential to impact the mink industry, especially in North America, and improve the overall performance of the mink industry.

Genetic characteristics of human parainfluenza viruses 1-4 associated with acute lower respiratory tract infection in Chinese children, during 2015-2021.

Human parainfluenza viruses (HPIVs) are a significant cause of acute lower respiratory tract infections (ALRTIs) among young children and elderly individuals worldwide. The four types of HPIVs (HPIV1-4) can cause recurrent infections and pose a significant economic burden on health care systems globally. However, owing to the limited availability of complete genome sequences, the genetic evolution of these viruses and the development of vaccines and antiviral treatments are hampered. To address this issue, this study utilized next-generation sequencing to obtain 156 complete genome sequences of HPIV1-4, which were isolated from hospitalized children with ALRTIs in six regions of China between 2015 and 2021. This study revealed multiple clades, lineages, or sublineages of HPIVs circulating in mainland China, with a novel clade D of HPIV1 identified as geographically restricted to China. Moreover, this study identified the endemic dominant genotype of HPIV3, lineage C3, which has widely spread and continuously circulated in China. Bioinformatic analysis of the genome sequences revealed that the proteins of HPIV3 possessed the most variable sites, with the P protein showing more diversity than the other proteins among all types of HPIVs. The HN proteins of HPIV1-3 are all under negative/purifying selection, and two amino acid substitutions in the HN proteins correspond to known mAb neutralizing sites in the two HPIV3 strains. These findings provide crucial insights into the genetic diversity and evolutionary dynamics of HPIVs circulating among children in China and may facilitate research on the molecular diagnosis, vaccine development, and surveillance of HPIVs.IMPORTANCEPhylogenetic analysis revealed the prevalence of multiple clades, lineages, or sublineages of human parainfluenza viruses (HPIVs) circulating in mainland China. Notably, a unique evolutionary branch of HPIV1 containing only Chinese strains was identified and designated clade D. Furthermore, in 2023, HPIV3 strains from Pakistan and Russia formed a new lineage within clade C, named C6. The first HPIV4b sequence obtained in this study from China belongs to lineage C2. Evolutionary rate assessments revealed that both the HN and whole-genome sequences of HPIV3 presented the lowest evolutionary rates compared with those of the other HPIV types, with rates of 6.98E-04 substitutions/site/year (95% HPD: 5.87E-04 to 8.25E-03) and 5.85E-04 substitutions/site/year (95% HPD: 5.12E-04 to 6.62E-04), respectively. Recombination analysis revealed a potential recombination event in the F gene of an HPIV1 strain in this study. Additionally, all the newly obtained HPIV1-3 strains exhibited negative selection pressure, and two mutations were identified in the HN protein of two HPIV3 strains at monoclonal antibody-binding sites.

Whole-genome sequencing identifies functional genes for environmental adaptation in Chinese sheep

Sheep (Ovis aries), among the first domesticated species, are now globally widespread and exhibit remarkable adaptability to diverse environments. In this study, we perform whole-genome sequencing of 266 animals from 18 distinct Chinese sheep populations, each displaying unique phenotypes indicative of adaptation to varying environmental conditions. Integrating 131 environmental factors with single nucleotide polymorphism variations, we conduct a comprehensive genetic-environmental association analysis. This analysis identifies 35 key genes likely integral to the environmental adaptation of sheep. The functions of these genes include fat tail formation (HOXA10, HOXA11, JAZF1), wool characteristics (FER, FGF5, MITF, PDE4B), horn phenotypes (RXFP2), reproduction (HIBADH, TRIM71, C6H4orf22), and growth traits (ADGRL3, TRHDE). Notably, we observe a significant correlation between the frequency of missense mutations in the PAPSS2 and RXFP2 genes and variations in altitude. Our study reveals candidate genes for adaptive variation in sheep and demonstrates the diversity in the ways sheep adapt to their environment.

Impact of a random TN5 mutation on endoglucanase secretion in ruminal cellulolytic Escherichia coli

ObjectiveMost protein secretion systems are found in gram-negative bacteria, but the mechanism of endoglucanase (BcsZ) secretion in Escherichia coli (E. coli) remains unclear. MethodsIn this study, we used JBZ-DH5α (which overexpresses BcsZ on the E. coli DH5α genome) as the initial strain. A mutant library was created by randomly inserting the TN5 transposon into the genome, and mutants with reduced transparent circles were identified on Congo red plates. The insertion sites of transposons in the genome were determined through whole-genome sequencing. ResultsThe results revealed that the genes rnc, lon, and suhB, which encode RNC-ribonuclease III (RNC), LON-protease (LON), and SuhB-inositol phosphatase (SuhB), respectively, were disrupted. BcsZ secretion decreased in E. coli DH5α when the lon, rnc, or suhB genes were deleted, but the overexpression of these genes restored their secretion levels. ConclusionThese findings suggest that the lon, rnc, and suhB genes play a role in BcsZ secretion in E. coli, potentially enhancing our knowledge of BcsZ secretion and offering a strategy to increase protein secretion in E. coli as a cell factory.

Exploring pangenomic diversity and CRISPR-Cas evasion potential in jumbo phages: a comparative genomics study.

Jumbo phages are large bacterial viruses known for more than 50 years. However, only in recent years, a significant number of complete genome sequences of jumbo phages have become available. In this study, we employed comparative genomic approaches to investigate the genomic diversity and genome protection capabilities of the 331 jumbo phages. Our findings revealed that jumbo phages exhibit high genetic diversity, with only a few genes being relatively conserved across jumbo phages. Interestingly, our data suggest that jumbo phages employ yet-to-be-identified strategies to protect their DNA from the host immune system, such as CRISPR-Cas.

Complete genome sequence of an umbravirus from white snakeroot (Ageratina altissima).

Complete genome sequencing of a virus from a white snakeroot plant (Ageratina altissima (L.) King & H. Rob.) collected in the Great Smoky Mountains National Park, USA, revealed a quadricistronic organization resembling that of umbraviruses. ORFs 1 and 2 are putatively translated via a -1 ribosomal frameshift mechanism as a single polypeptide with a role in viral replication, whereas the 3'-proximal and extensively overlapping ORFs 3 and 4 code for proteins involved in long distance trafficing andcell-to-cell movementwithin the host. Sequence comparisons and phylogenetic analysis strongly suggested that this virus is a previously undescribed member of the genus Umbravirus (family Tombusviridae), for which the name "white snakeroot virus A" (WSVA) is proposed. In addition, we identified and initiated characterization of its possible helper virus, a putative new member of the genus Luteovirus.

COMPARISON OF COMPUTATIONAL METHODS FOR ANALYSING 16S rRNA SEQUENCING DATA PRODUCED WITH THE USE OF SHORT AND LONG READS

This research is driven by the importance of identifying species in microbial community for use in medical testing. Conventional methods including culturing, biochemical assays are often limited to the culturable organisms and do not provide information about relative abundance of different microbial taxa within a community. Comparative analysis of metagenomic programs method was used to identify the most appropriate option for long-read 16S rRNA data. Since the development of high-throughput sequencing technologies, novel methods of identifying species include taxonomic profiling of 16S rRNA. 16S rRNA gene contains variable regions (V1-V9) that are specific to different taxonomic groups. Illumina short-read sequencing technology provides read lengths 2*251 base pairs (bp) for paired-end sequencing, while fraction of 16S rRNA gene is around 1500 bp, thus reads can successfully cover 2 of 9 regions (300-400 bp), but not the whole gene. The Oxford Nanopore Technology (ONT) representing long-read sequencing or so-called 3rd generation sequencing is much more applicable for 16S rRNA profiling, as it can provide reads with length 1500 bp (exactly in line with the length of 16S rRNA gene). Extended read length provides high alignment quality making the downstream taxonomy classification and relative abundance calculation much easier and relevant. The samples from endometrium of uterus were studied in the first part of the research. Endometrial microbiome composition and balance can play an important role in the success of in vitro fertilization (IVF). Taxonomical classification was tested with NCBI, Silva databases. NCBI database consists of full genomes of Bacteria, Archaea and Eukaryote, Silva contains curated and annotated sequences of only small subunit ribosomal RNA of these domains. Among the enormous collection of metagenome classifiers there were selected three particular programs applying completely different algorithms: Emu, PathoScope and Kraken2. The comparative process was divided in three phases. In the first one, pipelines were run with three programs and 11 samples using Silva and NCBI databases, the results were compared both quantitively and visually. In the second phase, the results were compared to the short-read taxonomical classification, the discrepancies were analyzed and questions were put together for further investigation. During the third phase pipelines with best performed programs were run on sample with acknowledged microbial diversity and following metrics were assessed and visualized: True positive, False positive, Precision, Recall, F1-score, L1-norm, L2-norm. Results: After the first phase it was revealed that Kraken2, Emu, PathoScope produce very similar results, while MetaScope discrepancies exceeded substantially 10% threshold for 70% of the reads. Additionally, Kraken2 annotated significantly more genera upon the threshold than other programs indicating its low specificity. Consequently, these two programs were excluded from further analysis. The second phase brought to the conclusion that short-read sequencing reports reveal only one prevailing genus in the community, while long-read establish 3 of them, thus it was assumed that short-read classification is much less sensitive. In the third phase it was determined that Emu and PathoScope have least deviation from reference value of abundances (ground truth) and they successfully annotate all the species introduced in the sample. Therefore, the main outcome of the research is the recommendation to use Emu and PathoScope programs for the purpose of identifying species using long-read data.

Improved inference of population histories by integrating genomic and epigenomic data.

With the availability of high-quality full genome polymorphism (SNPs) data, it becomes feasible to study the past demographic and selective history of populations in exquisite detail. However, such inferences still suffer from a lack of statistical resolution for recent, for example bottlenecks, events, and/or for populations with small nucleotide diversity. Additional heritable (epi)genetic markers, such as indels, transposable elements, microsatellites, or cytosine methylation, may provide further, yet untapped, information on the recent past population history. We extend the Sequential Markovian Coalescent (SMC) framework to jointly use SNPs and other hyper-mutable markers. We are able to (1) improve the accuracy of demographic inference in recent times, (2) uncover past demographic events hidden to SNP-based inference methods, and (3) infer the hyper-mutable marker mutation rates under a finite site model. As a proof of principle, we focus on demographic inference in Arabidopsis thaliana using DNA methylation diversity data from 10 European natural accessions. We demonstrate that segregating single methylated polymorphisms (SMPs) satisfy the modeling assumptions of the SMC framework, while differentially methylated regions (DMRs) are not suitable as their length exceeds that of the genomic distance between two recombination events. Combining SNPs and SMPs while accounting for site- and region-level epimutation processes, we provide new estimates of the glacial age bottleneck and post-glacial population expansion of the European A. thaliana population. Our SMC framework readily accounts for a wide range of heritable genomic markers, thus paving the way for next-generation inference of evolutionary history by combining information from several genetic and epigenetic markers.

Collaborative learning in the digital age: empowering tuberculosis researchers through virtual training.

Integrating whole genome sequencing (WGS) of the Mycobacterium tuberculosis complex into routine care, surveillance, and research in high tuberculosis burden settings remains challenging due to limited resources and skills. While technological platforms for scaling WGS are emerging, scaling wet lab and analytic components often depends on partnerships where such skills have been established. To address this, a virtual training program was developed. Over 12 weeks, 21 trainees from five Southern African institutes engaged in learning from curated theoretical content and interactive virtual meetings with experienced instructors. The training program, developed by a diverse team of experts in molecular biology, biomedical research, microbiology, and tuberculosis research, provided comprehensive coverage aligned with the latest advancements. Teaching strategies included interactive mentor-led sessions and real-time feedback, together with facilitated knowledge exchange and understanding. The virtual training program yielded several successes. Of note, trainees submitted three scientific articles for peer review, based on their acquired knowledge and its application in research. The program also fostered collaborations on Mycobacterium tuberculosis WGS among participants, showcasing the potential for networking and future joint projects. While the virtual training program encountered challenges related to the pandemic, limited resources, trainee engagement, and language barriers, these were creatively mitigated. To improve future training sessions, a platform assessing participant engagement and information retention is recommended. Wider collaborative efforts among experts and institutions in collating resources will lead to more comprehensive training programs. Addressing challenges such as internet connectivity issues and language barriers is crucial for ensuring inclusivity and enhancing the overall learning experience. In conclusion, the virtual training program successfully provided knowledge and skill training in WGS to trainees, leading to scientific article submissions and collaborations. Furthermore, content creators benefited from improved science communication and training opportunities.

Evaluation of the GenoType NTM-DR line probe assay for nontuberculous mycobacteria using whole genome sequences as reference standard

Pulmonary nontuberculous mycobacteria (NTM) disease is an emerging public health challenge that is especially problematic in people with cystic fibrosis (CF). Effective treatment depends on accurate species and subspecies identification and antimicrobial susceptibility status. We evaluated the GenoType NTM-DR VER 1.0 assay using biobanked NTM isolates with whole genome sequence (WGS) data and control isolates (total n=285). Species and subspecies detection sensitivity and specificity were 100 % for all species and subspecies except for two subspecies of M. intracellulare, that demonstrated a small degree of discrepant identification between M. intracellulare subspecies intracellulare and subspecies chimaera. All antimicrobial resistance markers were identified with 100 % sensitivity and specificity.We conclude that the GenoType NTM-DR assay offers a rapid and accurate option for identifying the most frequently encountered pathogenic NTM taxa and drug resistance markers.SUPPORT: Colorado CF Research Development Program and Colorado CF National Resource Centers funded by the Cystic Fibrosis Foundation, NJH Advanced Diagnostics Laboratories, Colorado Advanced Industries Accelerator Grant

First report of a carbapenem-resistant Aeromonas veronii environmental isolate in the United States co-harboring two carbapenemase genes

ObjectivesThe spread of carbapenem-resistant bacteria (CRB), and especially carbapenemase-producing CRB, is a global public health threat. Among them, Aeromonas species are of increasing concern because these emerging opportunistic pathogens are widespread in the environment and have increasingly been found to be resistant to carbapenems. The aim of this study was to investigate the genome and carbapenem-resistance determinants of Aeromonas veronii SS-M2-3, a highly carbapenem-resistant, carbapenemase-producing, river isolate from California (U.S). MethodsWe first used disk diffusion assays to characterize the susceptibility profile to carbapenems and other antibiotics of A. veronii SS-M2-3. We next used whole-genome sequencing using the Illumina platform and bioinformatics analysis to characterize the resistome of this isolate and identify its carbapenemase genes. ResultsA. veronii SS-M2-3 was resistant to all carbapenems tested and amoxicillin-clavulanic acid, whereas it was sensitive cefotaxime and all non-β-lactam antibiotics tested. Whole genome sequencing of this isolate revealed a complex resistome that included multidrug efflux pump genes and three chromosomal β-lactamase genes. These three genes encoded for highly conserved variants (82 to 97% amino acid identity) of the ChpA3 subclass B2 metallo-carbapenemase, OXA-12 class D carbapenemase, and the FOX-2 class C β-lactamase. This is the first report of an environmental A. veronni isolate from the U.S. co-harboring two carbapenemase genes. ConclusionsThese findings reveal that natural aquatic environments in the U.S. represent an underappreciated reservoir of carbapenem-resistant Aeromonas veronii isolates that can carry multiple carbapenemase genes.

The relationship between KL, ST types and virulence of the mucoid Acinetobacter baumannii

BackgroundAcinetobacter baumannii (A. baumannii) has become a significant nosocomial pathogen globally over the past decade due to the increasing prevalence of antibiotic-resistant isolates. The formation of the mucoid phenotype is a crucial adaptive defense response to external pressure, but the phenotypic and genotypic characteristics and their relationship with sequence types (ST) and K locus (KL) types remain unclear. MethodsIn this study, we screened a total of 736 A. baumannii isolates, from which we identified and characterized 13 mucoid isolates. The study explored the clinical characteristics of patients with mucoid isolates, investigated the mucoid phenotype, performed capsule observation, quantified capsule production, and assessed antimicrobial susceptibility. Subsequently, whole-genome sequencing (WGS) was used to analyze the sequence types (ST), loci for capsular polysaccharide (KL), antibiotic resistance genes, virulence genes, and core-genome single-nucleotide polymorphisms (SNPs). Additionally, the virulence of all mucoid strains was evaluated through serum resistance assay, biofilm-forming assay, and Galleria mellonella survival assay. ResultsAll mucoid A. baumannii isolates were found to be encapsulated and extremely drug-resistant. Among patients infected with these isolates, 92.3% had pulmonary infections, and the 30-day mortality rate was 61.5%. The analysis revealed that not all strains are highly virulent. Whole-genome sequencing (WGS) identified the sequence types as ST136, ST208, ST381, ST195, and ST281, and the capsular types as KL77, KL7, KL33, KL2, and KL3. The ST208 and KL7 isolates exhibited higher virulence and greater biofilm formation, with KL7 isolates also showing higher capsule production. Despite these differences, no significant variations in virulence genes were observed among the mucoid isolates, except for biofilm-associated and quorum-sensing genes. The highly virulent ST208/KL7 strains (AB276, AB313, and AB552) lacked biofilm-associated genes (csuA/BABCDE), indicating these genes do not directly cause differences in biofilm formation. ConclusionThe mucoid A. baumannii isolates were extensively drug-resistant, and infections caused by these isolates could lead to higher mortality. However, not all strains had high virulence, with variations likely related to specific sequence types (ST) and K locus (KL) types.