Whole Genome Sequencing Research Articles

Genome sequencing analysis of the pncA, rpsA and panD genes responsible for pyrazinamide resistance of Mycobacterium tuberculosis from Indonesian isolates.

Developing the most suitable treatment against tuberculosis based on resistance profiles is imperative to effectively cure tuberculosis patients. Whole-genome sequencing is a molecular method that allows for the rapid and cost-effective detection of mutations in multiple genes associated with anti-tuberculosis drug resistance. This sequencing approach addresses the limitations of culture-based methods, which may not apply to certain anti-TB drugs, such as pyrazinamide, because of their specific culture medium requirements, potentially leading to biased resistance culture results. Thirty-four M. tuberculosis isolates were subcultured on a Lowenstein-Jensen medium. The genome of these bacteria was subsequently isolated using cetyltrimethylammonium bromide. Genome sequencing was performed with Novaseq Illumina 6000 (Illumina), and the data were analysed using the GenTB and Mykrobe applications. We also conducted a de novo analysis to compare the two methods and performed mutation analysis of other genes encoding pyrazinamide resistance, namely rpsA and panD. The results revealed mutations in the pncA gene, which were identified based on the databases accessed through GenTB and Mykrobe. Two discrepancies between the drug susceptibility testing and sequencing results may suggest potential instability in the drug susceptibility testing culture, specifically concerning PZA. Meanwhile, the results of the de novo analysis showed the same result of pncA mutation to the GenTB or Mykrobe; meanwhile, there were silent mutations in rpsA in several isolates and a point mutation; no mutations were found in the panD gene. However, the mutations in the genes encoding pyrazinamide require further and in-depth study to understand their relationship to the phenotypic profile. Compared to the conventional culture method, the whole-genome sequencing method has advantages in determining anti-tuberculosis resistance profiles, especially in reduced time and bias.

Integration of Whole-Genome Sequencing with ddPCR Kit for Detection of Omicron Subvariants in Wastewater in the Upper Peninsula of Michigan

Integration of Whole-Genome Sequencing with ddPCR Kit for Detection of Omicron Subvariants in Wastewater in the Upper Peninsula of Michigan

Evaluation of whole genome sequencing utility in identifying driver alterations in cancer genome

In cancer genome analysis, identifying pathogenic alterations and assessing their effects on oncogenic processes is important. Although whole exome sequencing (WES) can effectively detect such changes, driver alterations could not be identified in 27.8% of the cases, according to a previous study. The objectives of the present study were to evaluate the utility of whole genome sequencing (WGS) and clarify its differences with WES in terms of driver alteration detection. For this purpose, WGS analysis was conducted on 177 driverless WES samples, selected from 5,480 fresh frozen samples derived from 5,140 Japanese patients with cancer. These samples were selected as primary tumor, both WES and transcriptome profiling were performed, estimated tumor content of ≥ 30%, and no driver alterations were identified by WES. WGS identified driver and likely driver alterations in 68.4 and 22.6% of the samples, respectively. The most frequent alteration type was oncogene amplification, followed by tumor suppressor gene deletion and small variants located outside the coding region. In the remaining 9.0% of samples, no such signals were identified; therefore, further investigations are required. The current study clearly demonstrated the role and utility of WGS in identifying genomic alterations that contribute to tumorigenesis.

Full-genome characterization of Turkish Rose Yellow Vein Virus (RYVV) Isolates

Rose yellow vein virus (RYVV) is a viral agent that has been identified in roses in recent years and is known to exist in a very limited area in the world. However, in studies conducted in our country, the presence of the agent has been detected in different regions and provinces. Since direct chemical control of viral diseases is not possible as in other biotic diseases, molecular characterization of the agent forms the basis for possible resistance or gene silencing studies to be conducted in the coming years. Thus, 44 RYVV isolates previously identified in our country were used for full genome studies. In full genome studies, overlapping PCR method was used with 7 different species-specific primer pairs. The full genomes of 3 of these isolates were obtained. The full genomes of the other isolates could not be obtained. It was determined that the genome size of the 3 RYVV isolates whose full genomes were obtained was 9314 nucleotides. It was determined that the only RYVV isolate found in the GenBank and the Türkiye isolates also showed a high level of nucleotide sequence similarity. However, the fact that the entire genome of the other isolates could not be obtained and the ones obtained were amplified with primer pairs designed according to the single isolate in the GenBank database may be due to the presence of different strains of RYVV isolates. For this reason, it is thought that next-generation sequencing studies should be prioritized to obtain complete genome information in future studies.

Whole genome analysis of Pantoea species identified from sepsis patients in selected Ethiopian referral hospitals: emerging pathogens

BackgroundThe burden of sepsis worsens due to the continuation of emerging pathogens such as multidrug-resistant Pantoea species.MethodsA multicenter study was conducted between October 2019 and September 2020 at four hospitals located in central, southern, and northern parts of Ethiopia. A total of 1416 sepsis patients were recruited and blood cultures were performed. At each study site, positive cultures were characterized by their colony characteristics, gram stain, and conventional biochemical tests. All Pantoea species were identified using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOF) and subjected to whole genome sequencing (WGS) using Illumina HiSeq 2500. The phylogeny structure of Pantoea isolates was calculated using IQ-TREE v1.6.12 from single-nucleotide polymorphisms detected by Snippy v.4.6.0 and filtered by Gubbins v.2.3.4. Average nucleotide identity was estimated by using OrthoANI v.0.93.1 on Shovill v.1.1.0 assemblies. Antimicrobial resistance genes and plasmid replicons were detected using ARIBA v.2.14.6. Phylogenetic trees were visualized using iTOLv.6.5.2.ResultsMultiple Pantoea species include: P. dispersa (n = 19), P. septica (n = 1), and a novel Pantoea spp. (n = 1), were identified among sepsis patients. All P. dispersa isolates and the novel Pantoea species were isolated at Dessie Referral Hospital and displayed phylogenetic clonality, including the ubiquity of an IncM1 plasmid and identical antimicrobial resistance (AMR) gene profiles, encoding blaCTX−M−15, blaTEM−1D, blaSCO−1, and aac(3)-lla. The novel Pantoea spp. isolate harboured blaCTX−M−9 and blaTEM−1D and carried an IncN3 plasmid replicon. The P. septica was isolated at Tikur Anbessa Specialized Hospital in Addis Ababa and carried no detectable acquired AMR genes.ConclusionThe emerging Pantoea spp. carrying multiple AMR genes were identified from sepsis patients. Implementation of strong infection prevention strategies and building surveillance capacity with advanced bacteriology laboratories capable of identifying multidrug-resistant emerging pathogens is strongly recommended.

Genomic Analyses of Methicillin-Resistant Staphylococcus pseudintermedius from Companion Animals Reveal Changing Clonal Populations, Multidrug Resistance, and Virulence.

Staphylococcus pseudintermedius is part of the normal microbiota in dogs. Since 2006, an increase in multidrug-resistant clones of methicillin-resistant S. pseudintermedius has been reported, as well as zoonotic transmission. Longitudinal investigations into clonal population structures, antibiotic resistance patterns, and the presence of resistance and virulence genes are important tools for gaining knowledge of the mechanisms behind the emergence of such clones. We investigated 87% of all non-repetitive MRSP isolates from dogs and cats in Sweden over a ten-year period (n = 356). All isolates were subjected to staphylococcal chromosomal cassette mec identification, whole-genome sequencing, multi-locus sequence typing, and analyses of genomic relatedness, as well as investigation of phenotypical resistance patterns and the presence of antibiotic resistance genes and virulence genes. A considerable increase over time in the number of clonal lineages present was observed, indicating genomic diversification, and four clones became dominant: ST71, ST258, ST265, and ST551. In total, 96% of the isolates were multidrug-resistant. Statistically significant differences in resistance to several antibiotic classes between the four dominant clones were present. All isolates carried several virulence genes encoding factors associated with attachment, colonization, toxin synthesis, quorum sensing, antibiotic resistance, and immune evasion.

Identification and genetic characterization of five novel bat coronaviruses from Yunnan, China

BackgroundCoronaviruses (CoVs) represent a serious threat to human health and have become a major transmissible, endemic, and causative pathogen in humans; they represent a major health concern, given their ability to cause infectious diseases. Bats are natural hosts for diverse viruses. Many transmission events of CoVs and identification of multiple novel CoVs in bats has increased attention towards their capacity to serve as hosts for zoonotic viruses.ResultsIn this study, 61 bats from Yunnan Province were analyzed, identifying seven CoVs, including three α- and two β-CoVs with full-genome sequences. Among the five identified alpha-CoVs, four belong to the Decacovirus subgenus and one to the Minunacovirus subgenus. Two beta-CoVs were also identified, both belonging to the Sarbecovirus subgenus.The genetic structures revealed similarities to known strains such as HKU10 and SARS-CoV-2, along with novel findings such as the Minunacovirus subgenus CoV YJ3c/f and unique ORF patterns. Our results demonstrated that strain JCC9 has a unique recombination pattern and shows a higher binding affinity to civet and pangolin ACE2 receptors, then the HpJC8xc strain transmits and recombines between hosts (bats), indicating a potential risk of crossing the interspecies barrier and infecting other animals.ConclusionsThe CoVs detected in the bats studied in this research exhibit high diversity. Genomic analysis revealed that CoVs in bats undergo frequent recombination events. Furthermore, recombination patterns and evolutionary analyses suggest that alpha-CoVs are more prone to cross-species transmission across different bat families/genera, whereas beta-CoVs demonstrate host specificity and tend to co-evolve with their bat hosts.Our finding suggest that bats, as hosts of CoVs, be constantly monitored to prevent outbreaks of new infections caused by viruses passing across interspecies barriers, and consequently, viral diseases in humans or livestock.

The complete mitochondrial genome of the endangered Atlantic Pygmy Devil Ray, Mobula hypostoma (Bancroft, 1831), from Brazil.

Mobulidae is a monophyletic family within the Myliobatiformes that comprises pelagic species represented by manta and devil rays. Among the genus Mobula, the Atlantic Pygmy Devil Ray - Mobula hypostoma - is reported in coastal regions exclusively in tropical and subtropical Atlantic Ocean from 1 to 100m deep. In Brazil, M. hypostoma is one of the least studied Mobula species. It is regularly misidentified, especially as Mobula thurstoni, and is commonly listed as bycatch, in fishery inventories, or related to opportunistic sightings in the national territory. Here, we describe the complete nucleotide sequence of the mitochondrial genome (mitogenome) from Mobula hypostoma, which is 18,141bp in length and comprises 13 protein-coding, two ribosomal RNA, and 22 transfer RNA genes. The M. hypostoma mitochondrial genes organisation and mitochondrial genome length are similar to other Mobula species, and the phylogenetic reconstruction indicates M. hypostoma as closely related to Mobula munkiana. The Brazilian mitogenome of M. hypostoma is expected to be a valuable resource for molecular-based species identification, and evolutionary and phylogeography studies.

Pure wild forest reindeer (Rangifer tarandus fennicus) or hybrids? A whole-genome sequencing approach to solve the taxonomical status

AbstractIn Finland, the geographic distribution of domestic reindeer (Rangifer tarandus tarandus) and Finnish wild forest reindeer (Rangifer tarandus fennicus) partly overlap in the vicinity of the southern border of the reindeer herding area. Additionally, domestic reindeer are occasionally kept as pets within the distribution range of the wild forest reindeer. Hybridisation of these two subspecies is one of the major threats for the wild forest reindeer population. Concerns about potential hybridisation served as the catalyst also for this study, which we aimed to clarify the taxonomic status of presumed wild forest reindeer individuals intended as founder individuals for a reintroduction project. To do this, we resequenced genomes of four Rangifer tarandus individuals with unknown taxonomical status and investigated their ancestries by comparing the genomic data with the existing resequenced data of the Finnish domestic reindeer and Finnish wild forest reindeer. The genetic relationship investigations indicated that all individuals we analysed were pure wild forest reindeer, making them suitable as founder individuals for the reintroduction project. Thus, our study provided critical knowledge for practical conservation action, where it was essential to recognise each individual’s origin. In the future, it will also offer novel insights into the spread of native wild forest reindeer to new geographic regions in Finland. For subsequent studies, additional resequenced genomic data of Rangifer individuals will be needed to develop an ancestry information marker panel of single nucleotide polymorphisms for rapid and cost-effective identification of hybrid individuals of these subspecies.

A novel missense variant in the RELN gene in sheep with lissencephaly and cerebellar hypoplasia.

Lissencephaly and cerebellar hypoplasia (LCH) represents a spectrum of congenital developmental malformations of the cerebral cortex and cerebellum, mostly occurring as inherited conditions caused by variants in an increasingly recognized number of genes. LCH has been identified in three Dorset-cross lambs with congenital neurological signs in Australia. Lambs were unable to walk and had reduced vision, and one lamb developed a hypermetric gait and intention tremors. Grossly, the lambs had diffuse pachygyria with reduction in white matter, mild bilateral ventriculomegaly of the lateral ventricles, and a markedly hypoplastic cerebellum. Histologically, there was disorganization of neurons within the cerebral cortex and hippocampus. The cerebellar vermis had disorganized, thin, and hypocellular gray matter with frequent ectopic Purkinje cells, while identifiable folia were largely absent within the hemispheres. Luxol fast blue stain and glial fibrillary acidic protein, neuronal nuclear protein, synaptophysin, and neuron-specific enolase immunohistochemistry confirmed the thickened, disorganized cerebral cortical gray matter and reduced white matter. Within the cerebellum, immunohistochemistry demonstrated marked dysplasia. Whole-genome sequencing analysis and prediction of variant effects identified a missense variant of interest in the candidate gene reelin (RELN; NC_040255.1:g.50288685C>T; NM_001306121.1:c.7088G>A; NP_001293050.1:p.(R2363H)) with a deleterious Sorting Intolerant from Tolerant (SIFT) score. Sanger sequencing identified that the variant segregated with LCH disease in the 3 affected individuals, their sire, and 6 unaffected flock members. The NP_001293050.1: p.(R2363H) substitution is predicted to decrease the stability of the protein (ΔΔG = -1.55 kcal/mol). Pathological and genetic findings are consistent with previously described phenotypes of RELN variants in Churra sheep, dogs, and humans.

SNP-Based and Kmer-Based eQTL Analysis Using Transcriptome Data.

Traditional expression quantitative trait locus (eQTL) mapping associates single nucleotide polymorphisms (SNPs) with gene expression, where the SNPs are derived from large-scale whole-genome sequencing (WGS) data or transcriptome data. While WGS provides a high SNP density, it also incurs substantial sequencing costs. In contrast, RNA-seq data, which are more accessible and less expensive, can simultaneously yield gene expressions and SNPs. Thus, eQTL analysis based on RNA-seq offers significant potential applications. Two primary strategies were employed for eQTL in this study. The first involved analyzing expression levels in relation to variant sites detected between populations from RNA-seq data. The second approach utilized kmers, which are sequences of length k derived from RNA-seq reads, to represent variant sites and associated these kmer genotypes with gene expression. We discovered 87 significant association signals involving eGene on the basis of the SNP-based eQTL analysis. These genes include DYNLT1, NMNAT1, and MRLC2, which are closely related to neurological functions such as motor coordination and homeostasis, play a role in cellular energy metabolism, and function in regulating calcium-dependent signaling in muscle contraction, respectively. This study compared the results obtained from eQTL mapping using RNA-seq identified SNPs and gene expression with those derived from kmers. We found that the vast majority (23/30) of the association signals overlapping the two methods could be verified by haplotype block analysis. This comparison elucidates the strengths and limitations of each method, providing insights into their relative efficacy for eQTL identification.

Dissemination and characterization of Escherichia coli resistant to extended-cephalosporins in feedlot lambs: A two-year two-population study

Dissemination of antimicrobial resistance in food animals is a One Health concern, but sheep production has been overlooked. This study aimed to explore the dissemination of Escherichia coli resistant to extended-spectrum cephalosporins (ESC) in feedlot lambs. Two pens were sampled on two occasions, and carcasses and other mammals living around were also tested. E. coli were recovered and antibiotic resistance determined. blaCTX-M/CMY genes and their genetic localization were characterized. Whole genome sequencing (WGS) was performed to confirm clonal relationship. The most prevalent ESC-resistance genes in the 108 E. coli isolates were blaCTX-M-55 (53.7 %), blaCTX-M-2 (14.8 %) and blaCMY-2 (13.9 %). Most blaCTX-M-55 genes were found on the chromosome, but IncA/C, IncHI1, IncHI2 and IncF plasmids were also identified. Genetic diversity was observed even though ST6448 was by far the most frequent ST. WGS analysis showed high similarity among isolates recovered from feedlot lambs, animals in the surroundings and lambs’ carcasses, proving the clonal and plasmidic dissemination.

Bovine Lactoferrin Impairs in-vitro Growth of Neonatal Sepsis-causingEscherichia Coli Clinical Isolates

Abstract Background E. coli is a dominant cause of neonatal sepsis with no prevention currently available. Lactoferrin (LF) is an iron-sequestering antibacterial protein that has been shown to decrease all-cause neonatal late-onset sepsis. However, the impact of LF on impairing growth of neonatal E. coli septicemia isolates carrying various iron acquisition virulence genes has not been investigated. We aimed to determine the effects of bovine lactoferrin (bLF) on in vitro growth of neonatal E. coli septicemia strains and to determine the presence of iron-acquisition virulence genes in the isolates. Methods We tested six clinical E. coli strains obtained from blood of septic neonates, including the archetypal RS218 strain. Isolates represent the most frequent phylogroups (B2 and D) and multi-locus sequence types (ST95, ST69, and ST131) among neonatal E. coli septicemia strains. The nonpathogenic strain DH5α was used as control. Strains were grown in media containing 0, 0.1, 1.0, and 10 mg/mL bLF. Optical density (OD) readings were obtained every 30 min. for 20 h. Experiments were repeated 3 times. OD area under the curve (AUC) values were compared with ANOVA. Whole genome sequencing (WGS) was obtained to determine the presence of iron-acquisition virulence genes using VirulenceFinder v. 2.0.3 (J Clin Micro 2020. 58 (10):e01269-20). Results For all strains, the growth decrease with 10 mg/mL bLF compared to zero bLF was highly significant (p < 0.001) (Fig.1). Growth was also significantly reduced at 1 mg/mL for most strains (p ≤ 0.02). Growth decrease at 0.1 mg/mL was only significant for isolate SCB31. Growth suppression at 10 mg/mL was greatest in DH5α. WGS analyses showed that all pathogenic strains carried several iron acquisition virulence genes, with chuA (hemin receptor), fyuA (siderophore receptor) in irp2 (protein 2 peptide synthetase) and sitA (iron transport protein) found in all (Fig. 2). DH5α had no iron acquisition virulence genes. Conclusion Bovine LF is effective in impairing growth of neonatal sepsis-causing E. coli regardless of the presence of iron acquisition genes known to contribute to virulence. Future studies will examine additional isolates and the effect of greater bLF concentrations. These data have the potential to inform dosing regimens for additional in vivo studies to further assess bLF’s protective effects against neonatal sepsis. Figure 1. Comparison of growth curve data among E. coli strains. Optical density AUC values of six neonatal septicemia E. coli isolates growth with increasing concentrations of bovine lactoferrin. DH5α is a nonpathogenic control strain. Bars depict means and standard deviations for each condition tested. Asterisks indicate statistically significant differences compared to the 0 mg/mL bLF condition (*p < 0.001, **p < 0.05). Figure 2. Presence of iron acquisition virulence genes in E. coli isolates. WGS analyses detected several genes, which are indicated in black. White indicates absence of gene. ST, Sequence type; chuA, Outer membrane hemin receptor; fyuA, Siderophore receptor (yersiniabactin); iroN, Siderophore receptor; irp2, High-molecular-weight protein 2 nonribosomal peptide synthetase; iucC, Aerobactin siderophore synthetase; iutA, Ferric aerobactin siderophore receptor; sitA, Iron transport protein. *Multi-drug resistant isolate.

Molecular and Seroepidemiologic Characteristics ofStaphylococcus aureus in Children with Cystic Fibrosis

Abstract Background Cystic fibrosis (CF), through altered sodium and chloride transport across the CF transmembrane receptor (CFTR), is associated with poor mucus clearance, bacterial colonization of the lower airway, chronic pulmonary disease punctuated by acute exacerbations, and chronic inflammation. With increased use of CFTR modulators, changes in the CF pulmonary microbiome have been observed, including decreased prevalence of Pseudomonas colonization; however, overall S. aureus prevalence remains largely unchanged. An important remaining question is whether changes in the CF pulmonary microbiome, while not affecting the overall prevalence of S. aureus colonization, has led to changes in S. aureus diversity or the strain types that are most frequently recovered from children with CF. It is also unknown whether the humoral immune response to S. aureus is altered in the presence of CFTR modulator therapy. Methods Genomic DNA was extracted from S. aureus isolates obtained from two cohorts: a historic cohort, comprised of children not receiving CFTR modulators, and a modern cohort, comprised of children receiving CFTR modulator therapy. Multilocus sequence typing (MLST) and whole genome sequencing (WGS) were used to identify clonotype and characterize virulence factors of interest. Serum was obtained from participants and antibody titers to the extracellular toxins LukAB and LukED were measured. Results One hundred participants were enrolled: 50 in cohort 1 (historic) and 50 in cohort 2 (modern). Compared to cohort 1, isolates identified in cohort 2 were less likely to contain ACME and mecA; similarly, cohort 2 isolates were more likely to contain PVL, sek, and sak. The frequency of specific clonotypes changed over time, with increased prevalence of CC8 and decreased prevalence of CC1, CC5, CC15, and CC30 in cohort 2 compared to cohort 1 (Figure 1A). Serum antibody concentrations to LukAB, a conserved toxin across S. aureus, were similar across cohorts (Figure 1B). Conclusion Over time, the clonotypes responsible for S. aureus colonization in patients receiving CFTR modulators have changed. Despite changes in clonotypes and receipt of CFTR modulators, serum concentrations of LukAB-specific antibodies did not differ. These data suggest that staphylococcal colonization in patients with CF is dynamic over time and may be modified based on the receipt of CFTR modulator therapy. Figure 1. (A) S. aureus clonal complexes, over time, demonstrating a relative increase in CC8 between 2012 and 2022; (B) serum concentrations of LukAB antibodies from 2012 (historic) and 2022 (modern) cohorts. No significant difference was observed in median anti-LukAB concentrations.

Serotype Distribution and Antimicrobial Resistance of Salmonella Isolates from Poultry Sources in China.

Salmonella is an important zoonotic pathogen, of which poultry products are important reservoirs. This study analyzed the prevalence, antimicrobial resistance, and characterization of Salmonella from broiler and laying hen sources in China. A total of 138 (12.27%) strains of Salmonella were isolated from 1125 samples from broiler slaughterhouses (20.66%, 44/213), broiler farms (18.21%, 55/302), and laying hen farms (6.39%, 39/610). Multiplex PCR was used to identify the serotypes. Antibiotic susceptibility testing to a set of 21 antibiotics was performed and all strains were screened by PCR for 24 selected antimicrobial resistance genes (ARGs). In addition, 24 strains of Salmonella were screened out by whole-genome sequencing together with 65 released Salmonella genomes to evaluate phylogenetic characteristics, multilocus sequence typing (MLST), and plasmid carriage percentages. A total of 11 different serotypes were identified, with the dominance of S. Enteritidis (43/138, 31.16%), S. Newport (30/138, 21.74%), and S. Indiana (19/138, 13.77%). The results showed that S. Enteritidis (34.34%, 34/99) and S. Newport (51.28%, 20/39) were the dominant serotypes of isolates from broilers and laying hens, respectively. The 138 isolates showed the highest resistance to sulfisoxazole (SXZ, 100%), nalidixic acid (NAL, 54.35%), tetracycline (TET, 47.83%), streptomycin (STR, 39.86%), ampicillin (AMP, 39.13%), and chloramphenicol (CHL, 30.43%), while all the strains were sensitive to both tigacycline (TIG) and colistin (COL). A total of 45.65% (63/138) of the isolates were multidrug-resistant (MDR) strains, and most of them (61/63, 96.83%) were from broiler sources. The results of PCR assays revealed that 63.77% of the isolates were carrying the quinolone resistance gene qnrD, followed by gyrB (58.70%) and the trimethoprim resistance gene dfrA12 (52.17%). Moreover, a total of thirty-four ARGs, eighty-nine virulence genes, and eight plasmid replicons were detected in the twenty-four screened Salmonella strains, among which S. Indiana was detected to carry the most ARGs and the fewest plasmid replicons and virulence genes compared to the other serotypes. This study revealed a high percentage of multidrug-resistant Salmonella from poultry sources, stressing the importance of continuous monitoring of Salmonella serotypes and antimicrobial resistance in the poultry chain, and emergency strategies should be implemented to address this problem.

Somatic mosaicism in schizophrenia brains reveals prenatal mutational processes.

Germline mutations modulate the risk of developing schizophrenia (SCZ). Much less is known about the role of mosaic somatic mutations in the context of SCZ. Deep (239×) whole-genome sequencing (WGS) of brain neurons from 61 SCZ cases and 25 controls postmortem identified mutations occurring during prenatal neurogenesis. SCZ cases showed increased somatic variants in open chromatin, with increased mosaic CpG transversions (CpG>GpG) and T>G mutations at transcription factor binding sites (TFBSs) overlapping open chromatin, a result not seen in controls. Some of these variants alter gene expression, including SCZ risk genes and genes involved in neurodevelopment. Although these mutational processes can reflect a difference in factors indirectly involved in disease, increased somatic mutations at developmental TFBSs could also potentially contribute to SCZ.

Screening of novel narrow-spectrum benzofuroxan derivatives for the treatment of multidrug-resistant tuberculosis through in silico, in vitro, and in vivo approaches.

Tuberculosis remains a serious global health threat, exacerbated by the rise of resistant strains. This study investigates the potential of two benzofuroxan (Bfx) derivatives, 5n and 5b, as targeted treatments for MDR-TB using in silico, in vitro, and in vivo methodologies. In vitro analyses showed that Bfx compounds have significant activity against Mtb H37Rv, with Bfx 5n standing out with a MIC90 of 0.09 ± 0.04 μM. Additionally, their efficacy against MDR and pre-XDR strains was superior compared to commercial drugs. These Bfx compounds have a narrow spectrum for mycobacteria, which helps avoid dysbiosis of the gut microbiota, and they also exhibit high selectivity and low toxicity. Synergism studies indicate that Bfx derivatives could be combined with rifampicin to enhance treatment efficacy and reduce its duration. Scanning electron microscopy revealed severe damage to the morphology of Mtb following treatment with Bfx 5n, showing significant distortions in the bacillary structures. Whole-genome sequencing of the 5n-resistant isolate suggests resistance mechanisms mediated by the Rv1855c gene, supported by in silico studies. In vivo studies showed that the 5n compound reduced the pulmonary load by 3.0 log10 CFU/mL, demonstrating superiority over rifampicin, which achieved a reduction of 1.23 log10 CFU/mL. In conclusion, Bfx derivatives, especially 5n, effectively address resistant infections caused by Mtb, suggesting they could be a solid foundation for future therapeutic developments against MDR-TB.

Mycobacterium tuberculosis complex sample processing by mechanical lysis, an essential step for reliable whole genome sequencing

Mycobacterium tuberculosis complex (MTBC) whole genome sequencing (WGS) turnaround time and WGS success rates are highly influenced by DNA extraction protocols even from cultures. Efficient mycobacterial lysis is crucial for obtaining sufficient DNA from cultures to facilitate reliable genomic drug susceptibility prediction and accurate genotyping with WGS. We compared four DNA extraction protocols from BD BACTEC™ Mycobacterial Growth Indicator Tubes (MGIT) for WGS with a focus on the lysis step: protocol A) column-based protocol without mechanical lysis; protocol B) an adapted protocol including a bead beating step; protocol C) DNA extraction from primary received cultures using bead beating: and protocol D) DNA extraction from pre MGIT-positive (enriched) cultures. Protocol B increased DNA yield approximately 60-fold, and significantly improved the sequencing success rate. The increased yield also allowed DNA extraction from primary cultures with high success rates (protocol C). Additionally, by using pre-positive enriched MGIT cultures, we demonstrated that bead beating opens the possibility of reliable WGS up to five days before a MGIT tube would be flagged positive (protocol D). The most optimal bead beating-based DNA extraction was also evaluated for Nanopore sequencing. Shortening bead beating duration to 15 s resulted in longer read lengths (N50 from 1.4 kb to 2.6 kb) while still providing efficient lysis. Furthermore, AmpureXP bead beating-based DNA capture / purification proved to be as efficient as Qiagen column-based DNA extraction, further simplifying and shortening the DNA extraction protocol. Adding a mechanical lysis step to our routine MTBC DNA extraction protocol has allowed us to reduce the turnaround time while maintaining DNA quality sequencing success rates.

The pathogenic germline ETV4 P433L mutation identified in multiple primary lung cancer affect tumor stem-like property by Wnt/β-catenin pathway

The occurrence of multiple primary lung cancer (MPLC) has witnessed a significant surge in recent years within the Chinese population. MPLC is distinguished by its potential genetic susceptibility and notable genetic heterogeneity. Investigating the etiology of MPLC holds substantial clinical importance.The whole genome sequencing (WGS) and genome-wide linkage analysis were performed in a family affected by a dominant form of lung abnormalities. Specifically, five family members were diagnosed with MPLC, while nine members had pulmonary nodules and one normal member. To confirm the potential pathogenic germline mutations sites, Sanger sequencing was performed in an additional 162 MPLC family patients. Furthermore, molecular biology experiments were conducted to investigate the function and the mechanism of the identified pathogenic mutation site in lung cancer A549 and H322, both in vitro and in vivo. Linkage analysis revealed the presence of shared genomic regions among affected family members. Subsequent exome sequencing identified a deleterious variant within these linkage intervals, specifically a heterozygous mutation in ETS-oncogene transcription factors 4 (ETV4). This particular variant was found in affected family members at a rate of 13 out of 15 individuals. Furthermore, ETV4 P433L mutation could be detected in an additional MPLC family patients and mutation frequency was 3.7% (6 out of 162). The ETV4 P433L mutations site was introduced into lung cancer cell lines, resulting in altered migration and stem-like properties of the cancer cells. Further investigation revealed that the activation of the Wnt/β-catenin signaling pathway, which is associated with stemness, could be attributed to the presence of the ETV4 P433L mutation, suggesting its involvement in tumor promotion. A novel pathogenic germline mutation, ETV4 P433L, was identified in a dominant MPLC family, with a mutation rate of 3.7% among MPLC family patients. The ETV4 P433L mutation was found to impact the stem-like properties and migration of tumors through Wnt/β-catenin signaling pathway.

Complete genome sequences of Enterococcus casseliflavus strains ASE2 and ASE4 generated by PacBio sequencing.

Enterococcus casseliflavus strains ASE2 and ASE4 were isolated from the soil of a lettuce farm in Salinas, California, in 2020. Their complete genome sequences were generated by the PacBio Sequel II platform. They both consist of a single circular chromosome without plasmids.