Genome Sequencing Methods Research Articles

Recent advances in degradation of N,N-diethyl-3-toluamide (DEET)-an emerging environmental contaminant: a review.

N,N-Diethyl-3-toluamide (DEET) is a commonly used insect repellent, which acts as an organic chemical contaminant in water and considered as an emerging contaminant which has been observed worldwide. It gets discharged into the environment through sewage waste. The various methods have been used to degrade DEET, such as UV based, ozonation, photocatalytic degradation, and biodegradation (based on the metabolic activity of fungi and bacteria). However, less research has been done on the degradation of DEET by deploying nanoparticles. Therefore, biodegradation and nanotechnology-based methods can be the potential solution to remediate DEET from the environment. This review is an attempt to analyze the routes of entry of DEET into the atmosphere and its environmental health consequences and to explore physical, chemical, and biological methods of degradation. Furthermore, it focuses on the various methods used for the biodegradation of the DEET, including their environmental consequences. Future research is needed with the application of biological methods for the degradation of DEET. Metabolic pathway for biodegradation was explored for the new potent microbial strains by the application of physical, chemical, and microbial genomics; molecular biology; genetic engineering; and genome sequencing methods.

Abstract IA15: Single cell origins and targeting of genomic instability in breast cancers

Abstract Early whole genome sequencing studies have emphasised the different modes by which breast cancers can evolve. However, bulk NGS tissue analysis obscures mutations that are contemporaneous and/or lead to negative selection, and does not resolve clone specific metrics such as replication rate and fitness. We have developed single cell genome sequencing methods (Laks et al., Cell 2019) to identify large scale copy number-structural (CNA-SV) mutational processes that result in genomic shuffling through the duplication or deletion of chromosomal regions, obscured by bulk tissue analysis. This has revealed the existence and activity of distinct “foreground” mutational processes, i.e. mutational events in single mitoses that are observed directly, before the effects of purifying selection. We discovered that foreground processes are preferentially associated with background patterns of genome instability (Funnell et al., Nature 2022). We also discovered that CNA-SV mutational patterns are associated with relative sensitivity or resistance to platinum, a commonly used DNA targeting agent in TNBC and HGSOC. Our second recent discovery has been the association of CNA-SV mutations with clonal resistance to platinum in TNBC (Salehi et al., Nature 2021). Taken together, we now understand that ongoing CNA-SV mutations contribute to the evolution of fitness landscapes for DNA targeting therapeutics. In parallel work (Xu et al Nat. comms 2017) we have discovered stabilisers of folded DNA guanine stacked bases, G-quadruplexes (G4), to be synthetically lethal in contexts including HR and TMEJ that are underpinned by the single cell processes identified through single genome sequencing. To this end we have recently observed G4-ligand clinical activity in BRCA2 and PALB2 mutated cancers in a phase 1 trial (Hilton et al, Nature communication 2022). Citation Format: Samuel Aparicio. Single cell origins and targeting of genomic instability in breast cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr IA15.

Targeted genomic sequencing of avian influenza viruses in wetland sediment from wild bird habitats.

Diverse influenza A viruses (IAVs) circulate in wild birds, including highly pathogenic strains that infect poultry and humans. Consequently, surveillance of IAVs in wild birds is a cornerstone of agricultural biosecurity and pandemic preparedness. Surveillance is traditionally done by testing wild birds directly, but obtaining these specimens is labor intensive, detection rates can be low, and sampling is often biased toward certain avian species. As a result, local incursions of dangerous IAVs are rarely detected before outbreaks begin. Testing environmental specimens from wild bird habitats has been proposed as an alternative surveillance strategy. These specimens are thought to contain diverse IAVs deposited by a broad range of avian hosts, including species that are not typically sampled by surveillance programs. To enable this surveillance strategy, we developed a targeted genomic sequencing method for characterizing IAVs in these challenging environmental specimens. It combines custom hybridization probes, unique molecular index-based library construction, and purpose-built bioinformatic tools, allowing IAV genomic material to be enriched and analyzed with single-fragment resolution. We demonstrated our method on 90 sediment specimens from wetlands around Vancouver, Canada. We recovered 2,312 IAV genome fragments originating from all eight IAV genome segments. Eleven hemagglutinin subtypes and nine neuraminidase subtypes were detected, including H5, the current global surveillance priority. Our results demonstrate that targeted genomic sequencing of environmental specimens from wild bird habitats could become a valuable complement to avian influenza surveillance programs.IMPORTANCEIn this study, we developed genome sequencing tools for characterizing avian influenza viruses in sediment from wild bird habitats. These tools enable an environment-based approach to avian influenza surveillance. This could improve early detection of dangerous strains in local wild birds, allowing poultry producers to better protect their flocks and prevent human exposures to potential pandemic threats. Furthermore, we purposefully developed these methods to contend with viral genomic material that is diluted, fragmented, incomplete, and derived from multiple strains and hosts. These challenges are common to many environmental specimens, making these methods broadly applicable for genomic pathogen surveillance in diverse contexts.

Unveiling mechanisms for induced systemic resistance, resistance breeding and molecular marker‐assisted breeding against Phomopsis blight of Solanum melongena

AbstractPhomopsis vexans is one of the most destructive fungal pathogens associated with eggplant and currently poses a significant threat to eggplant production worldwide. The detrimental impact of P. vexans on eggplant yield has been extensively explored by various mycologists who have conducted thorough studies on the diversity, pathology and biological aspects of the pathogen. However, achieving enduring resistance or effective management has proven to be a challenge thus far. PCR‐based detection and molecular association of Phomopsis resistance use molecular markers to examine the potential for heterosis in various crosses, aiming for premium hybrids with genetic resistance and high‐yielding capabilities. The latest genome sequencing methods and availability of a wider range of genetic diversity has enabled the breeding of resistant varieties of eggplant. This review provides a detailed description on P. vexans including its epidemiology, dispersal methods, symptomology, colony characteristics, taxonomy and evolution of its strains. Different resistance breeding techniques including heterosis breeding, host plant resistance, identification of resistant sources, inheritance pattern for Phomopsis resistance, importance of grafting to impart resistance, significance of induced resistance, PCR‐based detection and molecular association of Phomopsis resistance are explained. Future approaches include molecular marker techniques such as genome‐wide association studies, sequence‐characterized amplified regions (SCAR), role of biotic inducers, pathogenesis‐related proteins and plant growth‐promoting rhizobacteria against Phomopsis blight of eggplant.

Identification of sex locus and a male-specific marker in blunt-snout bream (Megalobrama amblycephala) using a whole genome resequencing method

Fish sex is often associated with its economic traits, such as growth and body color. The mechanism of fish sex determination is always a hot spot for scientific research. Fish is one of the most diverse groups of vertebrates, and its sex determination mechanism is diverse and complex. Blunt-snout bream (BSB), belonging to Cyprinidae and the genus Megalobrama, is an important economic fish in China. Ordinary consumers are more inclined to buy male fish that does not carry eggs, hence the preparation of this all-male group of BSB meets the needs of the industry. In this study, we applied a whole genome resequencing approach to search sex-linked markers and better characterized sex determination in this fish. Our results confirmed that BSB uses a male heterogametic (XX/XY) system. Individuals from two populations including 10 males and 10 females were sequenced using a whole genomic sequencing method. A large potential sex differentiated region with approximately 6 Mb at the beginning of linkage group (LG) 9 of female BSB genome was identified using genome comparative analysis. This region contained a high density of male-biased genetic polymorphisms and harbored 275 genes, whereas reported sex-related genes didn't exist in this region. With the help of male genome, some male-biased sequences were identified using genome-wide associated analysis, and subsequently a male-specific marker was developed. Our results exhibited that BSB had a considerably large sex locus on LG9, which was likely sex chromosome of this species. Moreover, we developed a male-specific marker for BSB. Our study not only is of great practical significance to the development of sex-controlled breeding technology, but also is of theoretical significance to further explore the sex determination mechanism in fish.

Molecular characteristics and pathogenic mechanisms of KPC-3 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (ST23-K1).

This study aimed to comprehensively investigate hypervirulent carbapenem-resistant Klebsiella pneumoniae (CR-hvKP) in the Ningbo region. Importantly, we sought to elucidate its molecular characteristics and pathogenic mechanisms. This information will provide evidence-based insights for preventing and controlling nosocomial infections and facilitate improved clinical diagnosis and treatment in this region. 96 carbapenem-resistant Klebsiella pneumoniae strains were collected from the Ningbo region between January 2021 and December 2022. Whole genome sequencing and bioinformatic methods were employed to identify and characterize CR-hvKP strains at the molecular level. The minimum inhibitory concentrations (MICs) of common clinical antibiotics were determined using the VITEK-2 Compact automatic microbiological analyzer. Plasmid conjugation experiments evaluated the transferability of resistance plasmids. Finally, mouse virulence assays were conducted to explore the pathogenic mechanisms. Among the 96 strains, a single CR-hvKP strain, designated CR-hvKP57, was identified, with an isolation frequency of 1.04%. Whole-genome sequencing revealed the strain to be ST23 serotype with a K1 capsule. This strain harbored three plasmids. Plasmid 1, a pLVPK-like virulence plasmid, carried multiple virulence genes, including rmpA, rmpA2, iroB, iucA, and terB. Plasmid 2 contained transposable element sequences such as IS15 and IS26. Plasmid 3, classified as a resistance plasmid, harbored the bla KPC-3 carbapenem resistance gene. Mouse virulence assays demonstrated a high mortality rate associated with CR-hvKP57 infection. Additionally, there was a significant increase in IL-1β, IL-6, and TNF-α levels in response to CR-hvKP57 infection, indicating varying degrees of inflammatory response. Western blot experiments further suggested that the pathogenic mechanism involves activation of the NF-κB signaling pathway. This study confirms the emergence of hypervirulent CR-hvKP in the Ningbo region, which likely resulted from the acquisition of a pLVPK-like virulence plasmid and a bla KPC-3 resistance plasmid by the ST23-K1 type Klebsiella pneumoniae. Our findings highlight the urgent need for more judicious use of antibiotics to limit the emergence of resistance. Additionally, strengthening infection prevention and control measures is crucial to minimize the spread of virulence and resistance plasmids.

Circulating Tumor DNA Profiling Approach Based on In Silico Background Elimination Guides Chemotherapy in Nasopharyngeal Carcinoma.

Circulating tumor DNA (ctDNA) analysis increasingly provides a promising minimally invasive alternative to tissue biopsies in precision oncology. However, there are no ctDNA analysis approaches available in nasopharyngeal carcinoma (NPC) and current methods of ctDNA mutation profiling have limited resolution because of the high background noise and false-positive rate caused by benign variants in plasma cell-free DNA (cfDNA), majorly generated during clonal hematopoiesis. Although personalized parallel white blood cell genome sequencing suppresses the noise of clonal hematopoiesis variances, the system cost and complexity restrict its extensive application in clinical settings. We developed Matched WBC Genome sequencing Independent CtDNA profiling (MaGIC) approaches, which synergically integrated a ctDNA capturing panel for a hybrid capture cfDNA deep sequencing, in silico background elimination, and a reliable readout measurement. We profiled the ctDNAs of 80 plasma samples from 40 patients with NPC before and during chemotherapy by MaGICs. In addition, the public cfDNA sequencing data and The Cancer Genome Atlas project data were analyzed by MaGICs to evaluate their application in other scenarios of patient classification. The MaGIC version-2 has the ability to predict the chemosensitivity of patients with NPC with high accuracy by utilizing a single sample of liquid biopsy from each patient prior to a standardized treatment regimen. Moreover, both versions of MaGICs are of ideal performance in the diagnosis of patients with prostate cancer by liquid biopsy and prognosis prediction of multiple cancers by tissue biopsy. This study has the potential to enhance the sensitivity and expand the application scope of ctDNA detection, independently of other paired genome sequencing methods. As a result, it might further increase the clinical utilization of liquid biopsy based on ctDNA.

Abstract C047: Multiomics approaches to identify African-ancestry specific phenotypes of triple negative breast cancer

Abstract Despite recent convergence of breast cancer (BC) incidence among African American (AA) and European American (EA) women, BC mortality remains 40% higher among AA women. This disparity is driven partly by BC heterogeneity, where distinct subtypes have differing prognosis and treatment modalities, defined by presence of estrogen and/or progesterone receptor (HR+), overexpression of HER2, or lack of all three biomarkers (triple negative BC (TNBC)). TNBC is the most aggressive subtype, diagnosed as late-stage disease and lacking targeted therapeutic options. TNBC represents ~10% of annual diagnoses, however prevalence is significantly higher among AA women. Our work in the International Center for the Study of Breast Cancer Subtypes (ICSBCS) revealed significant association of west African (AFR) ancestry and TNBC, where we report that Ghanaian and AA women have increased frequency of TNBC, compared to Ethiopian and EA women, and that TNBC patients have higher percentages of quantified AFR ancestry. To characterize AFR ancestry-specific TNBC phenotypes, we have employed transcriptomics and whole genome sequencing (WGS) methods. Our recent publication from an AFR ancestry enriched TNBC transcriptomics cohort (n = 26) leveraged genetic ancestry to identify 600+ AFR and 2000+ AFR sub-continental ancestry associated genes. Pathway analysis showed enrichment of immune cell trafficking and indicates distinct AFR ancestry phenotypes that can be leveraged for future therapeutic opportunities. Recently, we have completed WGS on 80+ AFR ancestry patients with TNBC disease (n = 59) and HR+ disease (n = 23); representing AA (n = 71), Ethiopian (n = 8) and Ghanaian patients (n = 3). TP53 was the most mutated gene, more commonly mutated among TNBC cases (83%) compared to HR+ cases (35%) (p < 0.0001). HR+ had significantly more GATA3 (26%) and PIK3CA (30%) mutations compared to TNBC (0% and 9%, p < 0.05). Prevalence of COSMIC mutational signatures shows higher Signature 1 among HR+ cases, and higher Signature 3 among TNBC cases (both p < 0.05). Signature 3 is associated with DNA double-strand break repair and germline BRCA1/2 mutation status, where 9 TNBC cases have elevated homologous repair deficiency status. Our genomic work utilizes the reference genome, which is primarily sequenced from a single individual. With the African Pan Genome (APG), a set of 125,000+ contigs distinct from the reference genome, we have aligned RNAseq reads to the APG that failed to map to the reference genome. Successful APG alignment of a subset of reads indicates that genes are expressed from these distinct AFR sequences. We also identified CpG sites in contigs with gene expression, showing AFR ancestry specific regulatory regions in the previously unmapped genome. We are working to determine the significance of these in the tumor microenvironment. This work represents a comprehensive, AFR ancestry enriched cohort with multiple genomic data approaches that will allow us to further characterize TNBC disease and define actionable biomarkers and targets for women of African ancestry. Citation Format: Rachel Martini, Max Chao, Timothy Chu, Brian Stonaker, Ishmael Kyei, Ernest Adjei, Mahteme Bekele, Kofi Gyan, Olivier Elemento, Nicolas Robine, John Carpten, Lisa Newman, Melissa Davis. Multiomics approaches to identify African-ancestry specific phenotypes of triple negative breast cancer [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C047.

Validation of an HIV whole genome sequencing method for HIV drug resistance testing in an Australian clinical microbiology laboratory.

Detection of HIV drug resistance (HIVDR) is vital to successful anti-retroviral therapy (ART). HIVDR testing to determine drug-resistance mutations is routinely performed in Australia to guide ART choice in newly diagnosed people living with HIV or in cases of treatment failure. In 2022, our clinical microbiology laboratory sought to validate a next-generation sequencing (NGS)-based HIVDR assay to replace the previous Sanger-sequencing (SS)-based ViroSeq. NGS solutions for HIVDR offer higher throughput, lower costs and higher sensitivity for variant detection. We sought to validate the previously described low-cost probe-based NGS method (veSEQ-HIV) for whole-genome recovery and HIVDR-testing in a diagnostic setting. veSEQ-HIV displayed 100% and 98% accuracy in major and minor mutation detection, respectively, and 100% accuracy of subtyping (provided > 1000 mapped reads were obtained). Pairwise comparison exhibited low inter-and intrarun variability across the whole-genome (Jaccard index [J] = 0.993; J = 0.972) and the Pol gene (J = 0.999; J = 0.999), respectively. veSEQ-HIV met all our pre-set criteria based on WHO recommendations and successfully replaced ViroSeq in our laboratory. Scaling-down veSEQ-HIV to a limited batch size and sequencing on Illumina iSeq. 100, allowed easy implementation of the assay into the workflow of a small sequencing laboratory with minimal staff and equipment and the ability to meet clinically relevant test turn-around times. As HIVDR-testing moves from SS- to NGS-based methods and new ART drugs come to market (particularly those with targets outside the Pol region), whole-genome recovery using veSEQ-HIV provides a robust, cost-effective and "future-proof" NGS method for HIVDR-testing.

DNMT3A Mutants Are Enriched in NPMc+ AML and Associated with Adverse Outcome in Childhood AML

Background: Somatic mutations in the DNA methyltransferase 3 alphagene (DNMT3A) are common in adult acute myeloid leukemia (AML), but are rare events in childhood AML (Ho, Ped Blood Cancer, 2011). Missense mutations occur most frequently in the catalytic domain at R882, resulting in loss of enzyme methylation activity and the ability of the enzyme to bind DNA. DNMT3Amutations are associated with adverse outcome in adults, while their impact is understudied in childhood AML. Our objective was to identify the prevalence of DNMT3A -mutated AML across the childhood age spectrum utilizing a large cohort of patients enrolled on pediatric trials and compare findings with adult cohorts. Methods: Pediatric patients treated on two consecutive COG trials with publicly available data were included (n=1607). Mutational profiling was performed by whole genome and transcriptome sequencing methods. Prevalence of DNMT3Amutations, co-occurring mutations, and outcomes (5-year event-free [EFS] and 5-year overall survival [OS]) were analyzed. Results: The prevalence of DNMT3Amutations amongst the entire childhood cohort was 1.2% (n=20/1607). DNMT3A mutations were absent in children <10 years with increased incidence up to 2.6% in patients >10yrs of age. All variants were missense, with the exception of one nonsense mutation and alterations at the 882 residue were the most common (70%). Most importantly, all but 4 DNMT3A mutations occurred in patients with NPM1 mutations (NPMc+; 80%). Conversely, DNMT3A mutations were seen in 9% of all NPM positive patients, demonstrating unique enrichment of this mutation within the NPM1 mutant cohort. Further, R882 mutations were seen in all but a single patient with the dual NPM/DNMT3A variant. It is notable that 11 patients harbored additional FLT3-ITD mutations (triple positive), which we have previously reported to be associated with adverse outcome in adults. In addition to mutations in NPM1 and FLT3, co-occurring variants with DNMT3A were also found in PTPN11 (35%), and WT1, IDH1/2 and MYC each at 10% frequency (Fig.A). Given the recent report of association of NPM1 mutation genotype and association of Type D mutation with adverse outcome (Pigazzi, ASH 2023), we evaluated NPM1 mutation genotype in those with cooperating DNMT3A mutation which demonstrated lack of association of DNMT3A mutations and Type D NPM1 mutations. Current risk classification in COG pediatric AML trials treat NPMc+ patients as favorable risk. Given the prevalence of DNMT3A mutations in NPMc+ patients we inquired whether DNMT3A mutations might modify outcome in this population. The 5 yr EFS estimates for NPMc+; patients with and without DNMT3A was 26.8% vs. 74.8% (p<0.0001; Fig.B) with a corresponding OS estimate at 5yrs of 60.3% vs. 82.2% (p=0.0169). We further concluded that presence or absence of FLT3-ITD does not impact the adverse outcome with similar 5 yr EFS for those with dual NPM1/DNMT3A or triple NPM1/DNMT3A/FLT3-ITD positive AML(20.0% vs. 30.3%respectively). These data are consistent with the adult DNMT3A data previously presented last year (Torabi; ASH 2022; abstract #306) regarding clinical implications of DNMT3A mutation in the setting of cooperating NPM1 mutation. Conclusion: Mutational profiling of children with AML identified DNMT3A mutations in adolescents and young adults and is enriched in those with NPMc+ mutations. As NPMc+ patients are considered to be favorable risk, knowledge of DNMT3A mutations would provide additional information for more precise risk stratification in childhood AML. Children with de novo AML and dual DNMT3A/NPM1 mutations remain at high risk of relapse and adverse outcome regardless of FLT3-ITD status.

Molecular and functional correction of a deep intronic splicing mutation in CFTR by CRISPR-Cas9 gene editing

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CFTR gene. The 10th most common mutation, c.3178-2477C>T (3849+10kb C>T), involves a cryptic, intronic splice site. This mutation was corrected in CF primary cells homozygous for this mutation by delivering pairs of guide RNAs (gRNAs) with Cas9 protein in ribonucleoprotein (RNP) complexes that introduce double-strand breaks to flanking sites to excise the 3849+10kb C>T mutation, followed by DNA repair by the non-homologous end-joining pathway, which functions in all cells of the airway epithelium. RNP complexes were delivered to CF basal epithelial cell by a non-viral, receptor-targeted nanocomplex comprising a formulation of targeting peptides and lipids. Canonical CFTR mRNA splicing was, thus, restored leading to the restoration of CFTR protein expression with concomitant restoration of electrophysiological function in airway epithelial air-liquid interface cultures. Off-target editing was not detected by Sanger sequencing of in silico-selected genomic sites with the highest sequence similarities to the gRNAs, although more sensitive unbiased whole genome sequencing methods would be required for possible translational developments. This approach could potentially be used to correct aberrant splicing signals in several other CF mutations and other genetic disorders where deep-intronic mutations are pathogenic.

Mabs, a suite of tools for gene-informed genome assembly

BackgroundDespite constantly improving genome sequencing methods, error-free eukaryotic genome assembly has not yet been achieved. Among other kinds of problems of eukaryotic genome assembly are so-called "haplotypic duplications", which may manifest themselves as cases of alleles being mistakenly assembled as paralogues. Haplotypic duplications are dangerous because they create illusions of gene family expansions and, thus, may lead scientists to incorrect conclusions about genome evolution and functioning.ResultsHere, I present Mabs, a suite of tools that serve as parameter optimizers of the popular genome assemblers Hifiasm and Flye. By optimizing the parameters of Hifiasm and Flye, Mabs tries to create genome assemblies with the genes assembled as accurately as possible. Tests on 6 eukaryotic genomes showed that in 6 out of 6 cases, Mabs created assemblies with more accurately assembled genes than those generated by Hifiasm and Flye when they were run with default parameters. When assemblies of Mabs, Hifiasm and Flye were postprocessed by a popular tool for haplotypic duplication removal, Purge_dups, genes were better assembled by Mabs in 5 out of 6 cases.ConclusionsMabs is useful for making high-quality genome assemblies. It is available at https://github.com/shelkmike/Mabs

Isolation and whole genome sequencing of a lipophilic anaerobic bacterium, a representative of the species complex <i>Corynebacterium tuberculostearicum</i>, from a tuberculosis focus

Background. The study of the lower respiratory tract microbiome has been actively developed inrecent years with the help of whole genome sequencing (WGS) methods. Due to this, it became clear that the nature of the lungs microbiota is very different from other microbial communities inhabiting the human body. One of the important directions in the study of pathological lungs biocenosis is the study of the role of the satellite microbiota of the tuberculosis focus. The aim of the work. To isolate and characterize oxygen-tolerant anaerobes from the necrotic contents of tuberculomas. Materials and methods. Biopsy material from 5 patients with pulmonary tuberculosis was obtained during a planned surgical treatment of tuberculoma. A pure culture was isolated from one sample during anaerobic cultivation. Lipase activity of strain was determined by plating on brain heart infusion agar (HIMEDIA, India) supplemented with 0.1 % Tween-80 and 10 mM of CaCl2. Antibiotic susceptibility was determined by RAPMYCO and SLOWMYCO of TREK Diagnostic Systems (Thermo Fisher Scientific, USA). DNA from the sediment of the broth culture was isolated by the CTAB chloroform method. Whole genome sequencing was performed on a DNBSeq-G400 NGS sequencer by Genomed (Russia). Results. Based on WGS results and phylogenetic analysis, the strain was identified as Corynebacterium kefirresidentii. The strain was characterized by high lipase activity and resistance only to Isoniazid, Ethionamide and Trimethoprim/Sulfamethoxazolin. Conclusion. The isolation of a lipophilic anaerobic representative of the Corynebacterium tuberculostearicum species complex from a tuberculous focus indicates a possible role of the non-tuberculous microbiota in the liquefaction of caseous necrosis. We assumed that in some cases, favorable conditions are created inside the tuberculous focus for the development of satellite anaerobic lipophilic microbiota.

A rapid and affordable amplicon-based method for next-generation genome sequencing of the infectious bursal disease virus

The infectious bursal disease virus (IBDV) causes a severe immunosuppressive disorder in young chickens. IBDV evolution resulted in the emergence of strains with divergent genetic, antigenic, and pathogenic characteristics. Genetic classification is typically performed by sequencing the coding region of the most immunogenic region of the viral protein 2 (VP2). Sequencing both double-stranded RNA genome segments is essential to achieve a more comprehensive IBDV classification that can detect recombinants and reassortments. Here, we report the development and standardization of a tiled PCR amplicon protocol for the direct and cost-effective genome sequencing of global IBDV strains using next-generation technology. Primers for tiled PCR were designed with adapters to bypass expensive and time-consuming library preparation steps. Sequencing was performed on Illumina MiniSeq equipment, and fourteen complete genomes of field strains were assembled using reference sequences. The PCR-enrichment step was used to obtain genomes from low-titer biological samples that were difficult to amplify using traditional sequencing. Phylogenetic analyses of the obtained genomes confirmed previous strain classification. By combining the enrichment methodology with massive sequencing, it is possible to obtain IBDV genomic sequences in a fast and affordable manner. This procedure can be a valuable tool to better understand virus epidemiology.

Comparative Study on Tuberculosis Drug Resistance and Molecular Detection Methods Among Different Mycobacterium Tuberculosis Lineages.

This study aims to compare drug resistance and detection efficacy across different Mycobacterium tuberculosis lineages, offering insights for precise treatment and molecular diagnosis. 161 strains of Mycobacterium tuberculosis (M.tb) were tested for drug resistance using Phenotypic Drug Susceptibility Testing (pDST), High-Resolution Melting analysis (HRM), and Whole Genome Sequencing (WGS) methods. The main focus was on evaluating the accuracy of different methods for detecting resistance to rifampicin (RIF), isoniazid (INH), and streptomycin (SM). Among the 161 strains of M.tb, 83.85% (135/161) were fully sensitive to RIF, INH, and SM according to pDST, and the rate of multidrug resistance was 4.35% (7/161). The drug resistance rates of lineage 2 M.tb to the three drugs (26/219, 11.87%) were significantly higher than those of non-lineage 2 M.tb (12/264, 4.45%) (P<0.05). Compared with pDST, WGS had a sensitivity of 100%, 94.12%, and 92.31% and a specificity of 100%, 99.31%, and 98.65% for RIF, INH, and SM, respectively, with no significant difference. The sensitivity of HRM for RIF, INH, and SM was 87.50%, 52.94%, and 76.92%, respectively, while the specificity was 96.08%, 99.31%, and 99.32%, respectively. The sensitivity of HRM for detecting INH resistance was significantly lower than that of pDST (P=0.039). Compared with HRM, WGS increased the sensitivity of RIF, INH, and SM by 12.50%, 41.18%, and 15.38%, respectively. There are significant differences in drug resistance rates among different lineages of M.tb, with lineage 2 having higher rates of RIF, INH, and SM resistance than lineages 3 and 4. The sensitivity of HRM is far lower than that of pDST, and currently, the accuracy of HRM is not sufficient to replace pDST. WGS has no significant difference in detecting drug resistance compared with pDST but can identify new anti-tuberculosis drug-resistant mutations, providing effective guidance for clinical decision-making.

Identification of ceftriaxone-resistant Neisseria gonorrhoeae FC428 clone and isolates harboring a novel mosaic penA gene in Chengdu in 2019–2020

BackgroundAntimicrobial resistance in gonorrhea has become a growing global public health burden. Neisseria gonorrhoeae isolates with resistance to ceftriaxone, the last remaining first-line option, represent an emerging threat of untreatable gonorrhea.MethodsA total of ten ceftriaxone-resistant N. gonorrhoeae FC428 isolates and two isolates harboring a novel mosaic penA-232.001 allele from 160 gonococcal isolates in Chengdu in 2019–2020 was described in the present study. Multilocus sequence typing (MLST) and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) were performed to characterize the isolates. Whole genome sequencing and maximum-likelihood method were performed to infer how the genetic phylogenetic tree of these isolates looks like. Recombination analysis was performed using the RDP4 software. This study was registered in the Chinese Clinical Trial Registry (ChiCTR2100048771, registration date: 20210716).ResultsThe genetic phylogeny showed that the ten FC428 isolates sporadically clustered into different phylogenetic clades, suggesting different introductions and local transmission of FC428. Two isolates showed close genetic relatedness to ceftriaxone-resistant clone A8806, which was only reported from Australia in 2013. Homologous recombination events were detected in penA between Neisseria gonorrhoeae and commensal Neisseria species (N. perflava and N. polysaccharea), providing evidence of commensal Neisseria species might serve as reservoirs of ceftriaxone resistance-mediating penA sequences in clinical gonococcal strains.ConclusionsOur results demonstrate further dissemination of FC428 in China and resurgence risks of sporadic ceftriaxone-resistant A8806 to become the next clone to spread.

Genetic variation in the immunoglobulin heavy chain locus shapes the human antibody repertoire

Variation in the antibody response has been linked to differential outcomes in disease, and suboptimal vaccine and therapeutic responsiveness, the determinants of which have not been fully elucidated. Countering models that presume antibodies are generated largely by stochastic processes, we demonstrate that polymorphisms within the immunoglobulin heavy chain locus (IGH) impact the naive and antigen-experienced antibody repertoire, indicating that genetics predisposes individuals to mount qualitatively and quantitatively different antibody responses. We pair recently developed long-read genomic sequencing methods with antibody repertoire profiling to comprehensively resolve IGH genetic variation, including novel structural variants, single nucleotide variants, and genes and alleles. We show that IGH germline variants determine the presence and frequency of antibody genes in the expressed repertoire, including those enriched in functional elements linked to V(D)J recombination, and overlapping disease-associated variants. These results illuminate the power of leveraging IGH genetics to better understand the regulation, function, and dynamics of the antibody response in disease.

In vitro modeling of isoniazid resistance mechanisms in Mycobacterium tuberculosis H37Rv.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, has been a global threat to human beings for several decades. Treating tuberculosis has become more difficult as the prevalence of drug-resistant tuberculosis has increased globally. Evidence suggests that the comprehensive landscape of resistance mechanisms in MTB is ambiguous. More importantly, little is known regarding the series of events connected to resistance mechanisms in MTB before exposure to anti-TB drugs, during exposure to the drugs, and finally, when the MTB becomes resistant after exposure, upon analyses of its genome. We used the wild-type strain of MTB (H37Rv) in an in vitro model for generating induced resistance using a sub-inhibitory concentration of isoniazid, and the generated resistance-associated variants (RAVs) were identified using the whole genome sequencing method. The detection of an inhA promoter mutation (fabG1-15C>T), which results in increased production of InhA protein, was found to be a major mechanism for developing resistance to isoniazid in the first place. We observed adaptation of MTB resistance mechanisms in high isoniazid stress by alteration and abolishment of KatG due to the detection of katG S315N, the common region of mutation that confers isoniazid resistance, along with katG K414N, katG N138S, and katG A162E. Furthermore, we detected the ahpC-72C>T and ahpC 21C>A mutations, but further investigation is needed to determine their role in compensating for the loss of KatG activity. This suggests that increased InhA production is the main mechanism where there are low levels of isoniazid, whereas the alteration of KatG was found to be utilized in mycobacterium with a high concentration of isoniazid. Our work demonstrates that this in vitro approach of generating induced resistance could provide clinically relevant information after the fabG1-15C>T mutation, which is the common mutation found in clinical isolates. Moreover, other mutations detected in this work can also be found in clinical isolates. These findings may shed light on the impact of isoniazid in generating RAV and the resistance mechanism scenario that mycobacterium used under various isoniazid-pressuring conditions. More research is needed to understand better the role of RAV and mechanical resistance events within the mycobacterium genome in promoting a promising drug prediction platform that could lead to the right treatment for patients with MDR-TB and XDR-TB.

Long-read-based single sperm genome sequencing for chromosome-wide haplotype phasing of both SNPs and SVs.

Although localized haploid phasing can be achieved using long read genome sequencing without parental data, reliable chromosome-scale phasing remains a great challenge. Given that sperm is a natural haploid cell, single-sperm genome sequencing can provide a chromosome-wide phase signal. Due to the limitation of read length, current short-read-based single-sperm genome sequencing methods can only achieve SNP haplotyping and come with difficulties in detecting and haplotyping structural variations (SVs) in complex genomic regions. To overcome these limitations, we developed a long-read-based single-sperm genome sequencing method and a corresponding data analysis pipeline that can accurately identify crossover events and chromosomal level aneuploidies in single sperm and efficiently detect SVs within individual sperm cells. Importantly, without parental genome information, our method can accurately conduct de novo phasing of heterozygous SVs as well as SNPs from male individuals at the whole chromosome scale. The accuracy for phasing of SVs was as high as 98.59% using 100 single sperm cells, and the accuracy for phasing of SNPs was as high as 99.95%. Additionally, our method reliably enabled deduction of the repeat expansions of haplotype-resolved STRs/VNTRs in single sperm cells. Our method provides a new opportunity for studying haplotype-related genetics in mammals.

Phenotypic and genotypic discrimination of Francisella tularensis ssp. holarctica clades

Francisella tularensis is the causative agent of tularemia, a zoonotic disease with a wide host range. F. tularensis ssp. holarctica (Fth) is of clinical relevance for European countries, including Germany. Whole genome sequencing methods, including canonical Single Nucleotide Polymorphism (canSNP) typing and whole genome SNP typing, have revealed that European Fth strains belong to a few monophyletic populations. The majority of German Fth isolates belong to two basal phylogenetic clades B.6 (biovar I) and B.12 (biovar II). Strains of B.6 and B.12 seem to differ in their pathogenicity, and it has been shown that strains of biovar II are resistant against erythromycin. In this study, we present data corroborating our previous data demonstrating that basal clade B.12 can be divided into clades B.71 and B.72. By applying phylogenetic whole genome analysis as well as proteome analysis, we could verify that strains of these two clades are distinct from one another. This was confirmed by measuring the intensity of backscatter light on bacteria grown in liquid media. Strains belonging to clades B.6, B.71 or B.72 showed clade-specific backscatter growth curves. Furthermore, we present the whole genome sequence of strain A-1341, as a reference genome of clade B.71, and whole proteomes comparison of Fth strains belonging to clades B.6, B.71 and B.72. Further research is necessary to investigate phenotypes and putative differences in pathogenicity of the investigated different clades of Fth to better understand the relationship between observed phenotypes, pathogenicity and distribution of Fth strains.