Whole Genome Sequencing Techniques Research Articles

Whole Genome Sequencing Analysis of Cellulose-degrading Bacterium DC11 Isolated from Silkworm Excrement and Characterization of Its Key Cellulase Gene ytoP: Running Head: Characterization of strain DC11 and its key gene ytoP

In recent years, the microbial degradation of cellulose has emerged in addressing environmental pollution and acting as a substitute for scarce animal feeds. This study conducted a whole-genome sequencing analysis of a cellulose-degrading bacterium known as Bacillus subtilis DC11, previously isolated from the silkworm excrement. A critical cellulase gene was identified, namely ytoP. The ytoP gene was successfully cloned and expressed in E.coli using genetic engineering. ytoP is a segment of the endoglucanase gene in Bacillus subtilis DC11 and was found to be 1074 bp in size and encoded 357 amino acids. This study effectively constructed the cellulase expression vector and achieved successful expression of the ytoP gene from strain DC11 in E. coli BL21 (DE3). SDS-PAGE electrophoresis revealed that the protein had an approximate size range of 40–50 KDa and a concentration of around 3.675 mg/mL. An assay of enzyme activity demonstrated that the purified protein, with a concentration of approximately 100 μg/mL, exhibited a maximum activity of 12.980 U/mL. Through the integration of whole-genome sequencing and genetic engineering techniques, the critical cellulase gene ytoP from Bacillus subtilis DC11 has been successfully cloned and expressed, achieving highly efficient cellulase production. This study lays the foundation for large-scale applications of microbial cellulose degradation in the future.

Molecular epidemiology of Brucella abortus isolated from the environment in Ningxia Hui autonomous region, China

Brucellosis is among the key zoonotic infectious diseases in China, and The Ningxia Hui Autonomous Region represents a major endemic area, and it is one of the main causes of poverty in the region due to illness. In Ningxia, there is substantial research on Brucella melitensis, studies on the molecular epidemiology of Brucella abortus are notably scarce. Consequently, this study aims to undertake pathogenic isolation and molecular epidemiological research on Brucella abortus isolated from the environment in Ningxia, providing insights and evidence to advance the prevention and control measures for brucellosis in the region. Building on traditional pathogenic detection methods, this research employs whole-genome sequencing(WGS) techniques and bioinformatics software to conduct a phylogenetic comparison of Ningxia strains and strains of Brucella abortus from various geographical origins. The results indicate that four Brucella abortus strains are classified as biovar 3 and MLST type ST2. It is shown that the local strains were closer phylogenetic relationships with strains from Asian and European countries. The presence of Brucella abortus in certain environmental sectors of Ningxia indicates a risk of transmission from the environment to animals and subsequently to humans. In conclusion, the Brucella abortus exists in some farming environments in Ningxia, and exists for a long time. Therefore, it is necessary to strengthen the monitoring of the disinfection effect of the farming environment to provide a basis for the forward movement of the gate of brucellosis prevention and control.

Development of a 1-step multiplex PCR assay for the detection of S. Enteritidis, S. Pullorum, S. Typhimurium, and S. Infantis associated with poultry production

Salmonellosis in poultry is detrimental to the advancement of the breeding industry and poses hazards to human health. Approximately 2,600 Salmonella varieties exist, among which S. Enteritidis, S. Pullorum, S. Typhimurium, and S. Infantis are prevalent serotypes in the poultry industry in recent years. They can also infect humans by contaminating poultry eggs and meat. Therefore, identifying these serotypes is crucial for successful preventive and control interventions. The White-Kauffmann-Le Minor scheme is time-consuming and requires expensive reagents. Whole-genome sequencing (WGS) and other molecular biology techniques require skilled technical staff. In comparison, the polymerase chain reaction (PCR) is more accurate, rapid, and inexpensive, thus proving suitable for widespread application in the poultry industry. Here, we selected 4 specific primers: lygD, mdh, ipaJ, and SIN_02055, which correspond to detecting S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Infantis, respectively. They were integrated into a 1-step multiplex PCR method. We optimized the PCR method by utilizing specificity test results to determine the optimal annealing temperature (57°C). The PCR method exhibited excellent sensitivity for genomic DNA and bacterial cultures. We used the developed method to determine 157 clinical Salmonella isolates from various stages of the poultry production chain. The results aligned with serotype data generated via WGS analysis, demonstrating the method's excellent accuracy. In conclusion, this study developed a 1-step multiplex PCR method that simultaneously identifies S. Enteritidis, S. Typhimurium, S. Pullorum, and S. Infantis, allowing routine mass detection in the grass-root poultry industry.

Whole-Genome Sequencing-Based Confirmatory Methods on RT-qPCR Results for the Detection of Foodborne Viruses in Frozen Berries

Accurate detection, identification, and subsequent confirmation of pathogens causing foodborne illness are essential for the prevention and investigation of foodborne outbreaks. This is particularly true when the causative agent is an enteric virus that has a very low infectious dose and is likely to be present at or near the limit of detection. In this study, whole-genome sequencing (WGS) was combined with either of two non-targeted pre-amplification methods (SPIA and SISPA) to investigate their utility as a confirmatory method for RT-qPCR positive results of foods contaminated with enteric viruses. Frozen berries (raspberries, strawberries, and blackberries) were chosen as the food matrix of interest due to their association with numerous outbreaks of foodborne illness. The hepatitis A virus (HAV) and human norovirus (HuNoV) were used as the contaminating agents. The non-targeted WGS strategy employed in this study could detect and confirm HuNoV and HAV at genomic copy numbers in the single digit range, and in a few cases, identified viruses present in samples that had been found negative by RT-qPCR analyses. However, some RT-qPCR-positive samples could not be confirmed using the WGS method, and in cases with very high Ct values, only a few viral reads and short sequences were recovered from the samples. WGS techniques show great potential for confirmation and identification of virally contaminated food items. The approaches described here should be further optimized for routine application to confirm the viral contamination in berries.

Abstract IA023: Spatial reconstruction and temporal inference of DNA methylation alterations in primary human colorectal cancer

Abstract Phenotypic plasticity is thought to assist immune evasion, metastasis, and therapeutic resistance in tumor evolution. Epigenetic mechanisms such as DNA methylation provide for a more easily acquired and flexible, yet semi-stable adaptation by the tumor cell than hardwired genetic changes. Cancer-associated gain of DNA methylation at promoter CpG islands or distal regulatory elements is associated with transcriptional silencing, whereas loss of DNA methylation is observed at late-replicating, lamina-attached regions of the genome, tracking with cumulative cell divisions. One of the most well-documented genomic features predisposing to DNA hypermethylation is Polycomb Repressive Complex (PRC) occupancy in precursor cells, consistent with an epigenetic block to cellular differentiation as an early or predisposing event in carcinogenesis. A central question is whether this epigenetic switch from reversible PRC repression to more stable DNA hypermethylation occurs before onset of clonal expansion, or whether it represents an ongoing process during tumor evolution. Clonal expansion is a pivotal characteristic of cancer, and is thought to be initiated by a genetic alteration in a key gatekeeper driver gene. However, not all normal cells appear to be susceptible to malignant transformation following such an event. Multiple lines of evidence suggest that epigenetic heterogeneity among normal cells may affect their cancer-initiating potential. We have developed a tumor processing pipeline to deeply and comprehensively map DNA methylation patterns in primary human colorectal cancer (CRC) and adjacent tissues, while preserving spatial information, enabling inference of the temporal order of molecular alterations. We employ a suite of technologies to map the spatial heterogeneity of DNA methylation patterns in primary human CRC. These include a deep single-cell whole-genome bisulfite sequencing technique that improves genomic coverage several-fold over existing published protocols, providing DNA methylation measurements at the majority of CpGs in the genome in individual tumor cells. We have also calibrated and validated an epigenetic clock that allows us to accurately measure the relative replicative history of individual cells. We infer phylogenetic relationships among subclones, and relate these to their spatial distribution in the primary tumor. We find that some tumors have high proportions of PRC-associated clonal hypermethylation, indicating that these hypermethylation events were already present prior to clonal expansion, possibly contributing to susceptibility to malignant transformation in the cell-of-origin, whereas other tumors appear to have active PRC-associated CpG island hypermethylation ongoing during tumor expansion, generating subclonal heterogeneity, which may influence post-therapeutic recurrence potential. Citation Format: Peter W. Laird, Nathan J. Spix, Hsiao-yun Milliron, Manpreet Kalkat, Emily Eugster, David Sokol, Emily Jung, Paula Nolte, Kelly K. Krzyzanowski, Toshinori Hinoue, Hui Shen. Spatial reconstruction and temporal inference of DNA methylation alterations in primary human colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr IA023.

An Emerging Foodborne Pathogen Spotlight: A Bibliometric Analysis and Scholarly Review of Escherichia coli O157 Research.

Foodborne infections pose a substantial global threat, causing an estimated 600 million illnesses and resulting in approximately 420,000 deaths annually. Among the diverse array of pathogens implicated in these infections, Escherichia coli (E. coli), specifically the O157 strain (E. coli O157), emerges as a prominent pathogen associated with severe outbreaks. This study employs a comprehensive bibliometric analysis and scholarly review focused on E. coli O157 research. The bibliometric analysis highlights the significant role played by the United States in the E. coli O157 research domain. Further exploration underscores the noteworthy contributions of the researcher Doyle MP, whose body of work, consisting of 84 documents and an impressive H-Index of 49, reflects their substantial impact in the field. Recent research trends indicate a discernible shift towards innovative detection methods, exemplified by the adoption of CRISPR-CAS and Loop-Mediated Isothermal Amplification. Moreover, high-throughput whole-genome sequencing techniques are gaining prominence for the expeditious analysis of pathogenic E. coli strains. Scientists are increasingly exploring antimicrobial agents, including phage therapy, to address the challenges posed by antibiotic-resistant E. coli strains, thereby addressing critical concerns related to multi-drug resistance. This comprehensive analysis provides vital insights into the dynamic landscape of E. coli O157 research. It serves as a valuable resource for researchers, policymakers, and healthcare professionals dedicated to mitigating E. coli O157 outbreaks and advancing global public health strategies.

Whole-genome sequencing and RNA sequencing analysis reveals novel risk genes and differential expression patterns in hepatoblastoma

Hepatoblastoma (HB) is an uncommon malignant liver cancer primarily affecting infants and children, characterized by the presence of tissue that resembling fetal hepatocytes, mature liver cells or bile duct cells. The primary symptom in affected children is abdominal lumps. HB constitutes approximately 28% of all liver tumors and two-thirds of liver malignancies in the pediatric and adolescent population. Despite its high prevalence, the underlying mechanism of HB pathogenesis remain largely unknown. To reveal the genetic alternations associated with HB, we conducted a comprehensive genomic study using whole-genome sequencing (WGS) and RNA sequencing (RNA-seq) techniques on five HB patients. We aimed to use WGS to identify somatic variant loci associated with HB, including single nucleotide polymorphisms (SNPs), insertions and deletions (Indels), and copy number variations (CNVs). Notably, we found deleterious mutation in CTNNB1, AXIN2 and PARP1, previously implicated in HB. In addition, we discovered multiple novel genes potentially associated with HB, including BRCA2 and GPC3 which require further functional validation to reveal their contributions to HB development. Furthermore, the American College of Medical Genetics and Genomics (ACMG) analysis identified the ABCC2 gene was the pathogenic gene as a potential risk gene linked with HB. To study the gene expression patterns in HB, we performed RNA-seq analysis and qPCR validation to reveal differential expression of four candidate genes (IGF1R, METTL1, AXIN2 and TP53) in tumors compared to nonneoplastic liver tissue in HB patients (P-Val < 0.01). These findings shed lights on the molecular mechanisms underlying HB development and facilitate to advance future personalized diagnosis and therapeutic interventions of HB.

Metagenomic Shotgun Sequencing Reveals Specific Human Gut Microbiota Associated with Insulin Resistance and Body Fat Distribution in Saudi Women.

(1) Background: Gut microbiota dysbiosis may lead to diseases such as insulin resistance and obesity. We aimed to investigate the relationship between insulin resistance, body fat distribution, and gut microbiota composition. (2) Methods: The present study included 92 Saudi women (18-25 years) with obesity (body mass index (BMI) ≥ 30 kg/m2, n = 44) and with normal weight (BMI 18.50-24.99 kg/m2, n = 48). Body composition indices, biochemical data, and stool samples were collected. The whole-genome shotgun sequencing technique was used to analyze the gut microbiota. Participants were divided into subgroups stratified by the homeostatic model assessment for insulin resistance (HOMA-IR) and other adiposity indices. (3) Results: HOMA-IR was inversely correlated with Actinobacteria (r = -0.31, p = 0.003), fasting blood glucose was inversely correlated with Bifidobacterium kashiwanohense (r = -0.22, p = 0.03), and insulin was inversely correlated with Bifidobacterium adolescentis (r = -0.22, p = 0.04). There were significant differences in α- and β-diversities in those with high HOMA-IR and waist-hip ratio (WHR) compared to low HOMA-IR and WHR (p = 0.02, 0.03, respectively). (4) Conclusions: Our findings highlight the relationship between specific gut microbiota at different taxonomic levels and measures of glycemic control in Saudi Arabian women. Future studies are required to determine the role of the identified strains in the development of insulin resistance.

Using random forest to predict antimicrobial minimum inhibitory concentrations of nontyphoidal Salmonella in Taiwan

Antimicrobial resistance (AMR) is a global health issue and surveillance of AMR can be useful for understanding AMR trends and planning intervention strategies. Salmonella, widely distributed in food-producing animals, has been considered the first priority for inclusion in the AMR surveillance program by the World Health Organization (WHO). Recent advances in rapid and affordable whole-genome sequencing (WGS) techniques lead to the emergence of WGS as a one-stop test to predict the antimicrobial susceptibility. Since the variation of sequencing and minimum inhibitory concentration (MIC) measurement methods could result in different results, this study aimed to develop WGS-based random forest models for predicting MIC values of 24 drugs using data generated from the same laboratories in Taiwan. The WGS data have been transformed as a feature vector of 10-mers for machine learning. Based on rigorous validation and independent tests, a good performance was obtained with an average mean absolute error (MAE) less than 1 for both validation and independent test. Feature selection was then applied to identify top-ranked 10-mers that can further improve the prediction performance. For surveillance purposes, the genome sequence-based machine learning methods could be utilized to monitor the difference between predicted and experimental MIC, where a large difference might be worthy of investigation on the emerging genomic determinants.

Role of epigenetics in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers, associated with poor survival outcomes. Lack of early diagnosis, resistance to conventional therapeutic treatments (including immunotherapy) and recurrence are some of the major hurdles in PDAC and contribute to its poor survival rate. While the risk of genetic predisposition to cancers is widely acknowledged and understood, recent advances in whole-genome and next-generation sequencing techniques have led to the acknowledgment of the role played by epigenetics, especially in PDAC. Epigenetic changes are heritable genetic modifications that influence gene expression without altering the DNA sequence. Epigenetic mechanisms (e.g., DNA methylation, post-translational modification of histone complexes and ncRNA) that result in reversible changes in gene expression are increasingly understood to be responsible for tumor initiation, development and even escape from immune surveillance. Our review seeks to highlight the various components of the epigenetic machinery that are known to be implicated in PDAC initiation and development and the feasibility of targeting these components to identify novel pharmacological strategies that could potentially lead to breakthroughs in PDAC treatment.

Dietary Polyphenols in Relation to Gut Microbiota Composition in Saudi Arabian Females.

Polyphenols may modulate gut microbiota; however, limited studies have examined this relationship relative to obesity. We aim to investigate the association between polyphenol intake and gut microbiota composition in relation to obesity indices among Saudi Arabian females. This study included 92 adults stratified by body mass index (BMI) into controls (BMI ≥ 18.5-24.9 kg/m2; n = 48) and cases (BMI ≥ 30.0 kg/m2; n = 44), and further divided into high and low polyphenol intake by median intake (252 mg/1000 kcal/day). Fecal samples were collected to analyze the gut microbiota composition via the whole-genome shotgun sequencing technique. Results showed that Flavonifractor plautii and Clostridium bolteae were positively correlated with polyphenol intake in the total sample (r = 0.22, p = 0.03; r = 0.28, p = 0.01, respectively). There were inverse correlations between Blautia wexlerae and polyphenol intake (r = -0.56, p &lt; 0.01) in the case group, and between Bacteroides thetaiotaomicron and polyphenol intake (r = -0.45, p = 0.03) in the control group. Those in the case group with low polyphenol intake, and those with high waist-to-hip ratio (WHR; ≥0.83), showed significantly lower alpha-diversity than those in the control group with normal WHR (&lt;0.83), (p &lt; 0.05). Findings suggest that polyphenols are correlated with specific bacteria and may play an important role in the modulation of gut microbiota and obesity management.

DNA methylation dynamics during germline development

DNA methylation plays essential homeostatic functions in eukaryotic genomes. In animals, DNA methylation is also developmentally regulated and, in turn, regulates development. In the past two decades, huge research effort has endorsed the understanding that DNA methylation plays a similar role in plant development, especially during sexual reproduction. The power of whole-genome sequencing and cell isolation techniques, as well as bioinformatics tools, have enabled recent studies to reveal dynamic changes in DNA methylation during germline development. Furthermore, the combination of these technological advances with genetics, developmental biology and cell biology tools has revealed functional methylation reprogramming events that control gene and transposon activities in flowering plant germlines. In this review, we discuss the major advances in our knowledge of DNA methylation dynamics during male and female germline development in flowering plants.

Whole-genome sequencing holds the key to the success of gene-targeted therapies.

Rare genetic disorders affect as many as 3%-5% of all babies born. Approximately 10,000 such disorders have been identified or hypothesized to exist. Treatment is supportive except in a limited number of instances where specific therapies exist. Development of new therapies has been hampered by at least two major factors: difficulty in diagnosing diseases early enough to enable treatment before irreversible damage occurs, and the high cost of developing new drugs and getting them approved by regulatory agencies. Whole-genome sequencing (WGS) techniques have become exponentially less expensive and more rapid since the beginning of the human genome project, such that return of clinical data can now be achieved in days rather than years and at a cost that is comparable to other less expansive genetic testing. Thus, it is likely that WGS will ultimately become a mainstream, first-tier NBS technique at least for those disorders without appropriate high-throughput functional tests. However, there are likely to be several steps in the evolution to this end. The clinical implications of these advances are profound but highlight the bottlenecks in drug development that still limit transition to treatments. This article summarizes discussions arising from a recent National Institute of Health conference on nucleic acid therapy, with a focus on the impact of WGS in the identification of diagnosis and treatment of rare genetic disorders.

Molecular Epidemiology of Multidrug-Resistant Pseudomonas aeruginosa Acquired in a Spanish Intensive Care Unit: Using Diverse Typing Methods to Identify Clonal Types.

The increasing number of infections from multidrug-resistant P. aeruginosa (MDRPA) has compromised the selection of appropriate treatment in critically ill patients. Recent investigations have shown the existence of MDRPA global clones that have been disseminated in hospitals worldwide. We aimed to describe the molecular epidemiology and genetic diversity of the MDRPA acquired by Intensive Care Units (ICU) patients in our hospital. We used phenotypic methods to define the MDRPA and molecular methods were used to illustrate the presence of carbapenemase encoding genes. To characterize the MDRPA isolates, we used MALDI-TOF biomarker peaks, O-antigen serotyping, and multi-locus sequence typing analyses. Our data show that the most widely distributed MDRPA clone in our ICU unit was the ST175 strain. These isolates were further investigated by the whole-genome sequencing technique to determine the resistome profile and phylogenetic relationships, which showed, as previously described, that the MDR profile was due to the intrinsic resistance mechanisms and not the carbapenemase encoding genes. In addition, this study suggests that the combination of environmental focus and cross-transmission are responsible for the spread of MDRPA clones within our ICU unit. Serotyping and MALDI-TOF analyses are useful tools for the early detection of the most prevalent MDRPA clones in our hospital. Using these methods, semi-directed treatments can be introduced at earlier stages and healthcare professionals can actively search for environmental foci as possible sources of outbreaks.

Single-base resolution methylomes of somatic embryogenesis in Theobroma cacao L. reveal epigenome modifications associated with somatic embryo abnormalities

Propagation by somatic embryogenesis in Theobroma cacao has some issues to be solved, as many morphologically abnormal somatic embryos that do not germinate into plants are frequently observed, thus hampering plant production on a commercial scale. For the first time the methylome landscape of T. cacao somatic embryogenesis was examined, using whole-genome bisulfite sequencing technique, with the aim to understand the epigenetic basis of somatic embryo abnormalities. We identified 873 differentially methylated genes (DMGs) in the CpG context between zygotic embryos, normal and abnormal somatic embryos, with important roles in development, programmed cell death, oxidative stress, and hypoxia induction, which can help to explain the morphological abnormalities of somatic embryos. We also identified the role of ethylene and its precursor 1-aminocyclopropane-1-carboxylate in several biological processes, such as hypoxia induction, cell differentiation and cell polarity, that could be associated to the development of abnormal somatic embryos. The biological processes and the hypothesis of ethylene and its precursor involvement in the somatic embryo abnormalities in cacao are discussed.

Abstract 925: A genetically engineered mouse model for carcinogenesis by the human enzyme APOBEC3B

Abstract Introduction: Advanced DNA sequencing technologies have revealed a substantial endogenous source of mutations in cancers, the human DNA-mutating enzyme APOBEC3B (A3B). This protein changes DNA cytosines into uracils (C-to-U), which can become “immortalized” in the genome as C-to-T or C-to-G mutations depending on how each uracil lesion is processed. However, mice lack analogous carcinogenic A3 enzymes, which makes a definitive cause-and-effect model of A3-induced carcinogenesis difficult to establish. Results: Using C57BL/6 mice, we have generated a novel genetically engineered mouse model (GEMM) expressing the human protein A3B, where it is driven constitutively from the Rosa26 locus in combination with the strong synthetic CAG promoter. Importantly, tumor-free survival data suggest that CAG A3B mice develop tumors at an increased rate compared to wild-type mice. These CAG A3B mice develop predominantly lymphoid neoplasms including B- and T-cell lymphomas and marked splenomegaly, and less frequently lung and liver cancers. Using immunohistochemical and whole-genome sequencing techniques, we have further characterized the tumors arising in these novel GEMMs. The above analyses thus far have revealed that A3B is actively driving tumor formation, as seen by an enrichment of canonical APOBEC-related mutations in these cancers. Conclusion: Overall, the CAG A3B mice are the first GEMMs demonstrating the potential for A3B to drive tumor formation in vivo, with a predilection towards blood malignancies. They will be an invaluable tool for characterizing A3B-driven mutational processes in an organism and testing novel therapeutic strategies against APOBEC-induced cancer initiation and progression. Citation Format: Cameron C. Durfee, Prokopios P. Argyris, Matthew C. Jarvis, Rena Levin-Klein, Emily K. Law, Reuben S. Harris. A genetically engineered mouse model for carcinogenesis by the human enzyme APOBEC3B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 925.

Detection of Antimicrobial Resistance, Pathogenicity, and Virulence Potentials of Non-Typhoidal Salmonella Isolates at the Yaounde Abattoir Using Whole-Genome Sequencing Technique.

One of the crucial public health problems today is the emerging and re-emerging of multidrug-resistant (MDR) bacteria coupled with a decline in the development of new antimicrobials. Non-typhoidal Salmonella (NTS) is classified among the MDR pathogens of international concern. To predict their MDR potentials, 23 assembled genomes of NTS from live cattle (n = 1), beef carcass (n = 19), butchers’ hands (n = 1) and beef processing environments (n = 2) isolated from 830 wet swabs at the Yaounde abattoir between December 2014 and November 2015 were explored using whole-genome sequencing. Phenotypically, while 22% (n = 5) of Salmonella isolates were streptomycin-resistant, 13% (n = 3) were MDR. Genotypically, all the Salmonella isolates possessed high MDR potentials against several classes of antibiotics including critically important drugs (carbapenems, third-generation cephalosporin and fluoroquinolone). Moreover, >31% of NTS exhibited resistance potentials to polymyxin, considered as the last resort drug. Additionally, ≤80% of isolates harbored “silent resistant genes” as a potential reservoir of drug resistance. Our isolates showed a high degree of pathogenicity and possessed key virulence factors to establish infection even in humans. Whole-genome sequencing unveiled both broader antimicrobial resistance (AMR) profiles and inference of pathogen characteristics. This study calls for the prudent use of antibiotics and constant monitoring of AMR of NTS.

Comparing BeadChip and WGS Genotyping: Non-Technical Failed Calling Is Attributable to Additional Variation within the Probe Target Sequence.

Microarray-based genomic selection is a central tool to increase the genetic gain of economically significant traits in dairy cattle. Yet, the effectivity of this tool is slightly limited, as estimates based on genotype data only partially explain the observed heritability. In the analysis of the genomes of 17 Israeli Holstein bulls, we compared genotyping accuracy between whole-genome sequencing (WGS) and microarray-based techniques. Using the standard GATK pipeline, the short-variant discovery within sequence reads mapped to the reference genome (ARS-UCD1.2) was compared to the genotypes from Illumina BovineSNP50 BeadChip and to an alternative method, which computationally mimics the hybridization procedure by mapping reads to 50 bp spanning the BeadChip source sequences. The number of mismatches between the BeadChip and WGS genotypes was low (0.2%). However, 17,197 (40% of the informative SNPs) had extra variation within 50 bp of the targeted SNP site, which might interfere with hybridization-based genotyping. Consequently, with respect to genotyping errors, BeadChip varied significantly and systematically from WGS genotyping, introducing null allele-like effects and Mendelian errors (<0.5%), whereas the GATK algorithm of local de novo assembly of haplotypes successfully resolved the genotypes in the extra-variable regions. These findings suggest that the microarray design should avoid polymorphic genomic regions that are prone to extra variation and that WGS data may be used to resolve erroneous genotyping, which may partially explain missing heritability.

Molecular Typing of Ukrainian Bacillus anthracis Strains by Combining Whole-Genome Sequencing Techniques

Anthrax is a recurrent zoonosis in the Ukraine with outbreaks occurring repeatedly in certain areas. For determining whether several Bacillus anthracis genotypes are circulating in this region, four strains from various sources isolated from different regions of the Ukraine were investigated. By combining long- and short-read next-generation sequencing techniques, highly accurate genomes were reconstructed, enabling detailed in silico genotyping. Thus, the strains could be assigned to the Tsiankovskii subgroup of the “TransEurAsia” clade, which is commonly found in this region. Their high genetic similarity suggests that the four strains are members of the endemic population whose progenitor was once introduced in the Ukraine and bordering regions. This study provides information on B. anthracis strains from a region where there is little knowledge of the local population, thereby adding to the picture of global B. anthracis genotype distribution. We also emphasize the importance of surveillance and prevention methods regarding anthrax outbreaks, as other studies predicted a higher number of cases in the future due to global warming.

Clinical, radiological, and molecular diagnosis of congenital pituitary diseases causing short stature

Short stature in children can be caused by congenital pituitary disorders involving at least one form of growth hormone deficiency. Clinical and radiological evaluations of the index case and family history assessments are essential to guide genetic diagnostic testing and interpret results. The first-line approach is panel testing of genes involved in pituitary development with variants known to be pathogenic in this context. It identifies a genetic cause in less than 10% of cases, however. Whole-exome and whole-genome sequencing techniques may provide original information but also raise new questions regarding the pathophysiological role of identified variants. These new tools can make genetic counselling more complex. The role of clinicians in these interpretations is therefore important. © 2022 French Society of Pediatrics. Published by Elsevier Masson SAS. All rights reserved.