Whole Genome Sequencing Methods Research Articles

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.

Association of microbiological factors with mortality in Escherichia coli bacteraemia presenting with sepsis/septic shock: a prospective cohort study

ObjectivesThis study aimed to determine the association of Escherichia coli microbiological factors with 30-day mortality in patients with bloodstream infection (BSI) presenting with a dysregulated response to infection (i.e. sepsis or septic shock). MethodsWhole-genome sequencing was performed on 224 E coli isolates of patients with sepsis/septic shock, from 22 Spanish hospitals. Phylogroup, sequence type, virulence, antibiotic resistance, and pathogenicity islands were assessed. A multivariable model for 30-day mortality including clinical and epidemiological variables was built, to which microbiological variables were hierarchically added. The predictive capacity of the models was estimated by the area under the receiver operating characteristic curve (AUROC) with 95% confidence intervals (CI). ResultsMortality at day 30 was 31% (69 patients). The clinical model for mortality included (adjusted OR; 95% CI) age (1.04; 1.02–1.07), Charlson index ≥3 (1.78; 0.95–3.32), urinary BSI source (0.30; 0.16–0.57), and active empirical treatment (0.36; 0.11–1.14) with an AUROC of 0.73 (95% CI, 0.67–0.80). Addition of microbiological factors selected clone ST95 (3.64; 0.94–14.04), eilA gene (2.62; 1.14–6.02), and astA gene (2.39; 0.87–6.59) as associated with mortality, with an AUROC of 0.76 (0.69–0.82). DiscussionDespite having a modest overall contribution, some microbiological factors were associated with increased odds of death and deserve to be studied as potential therapeutic or preventive targets.

Multiple introductions of monkeypox virus to Ireland during the international mpox outbreak, May 2022 to October 2023.

BackgroundMpox, caused by monkeypox virus (MPXV), was considered a rare zoonotic disease before May 2022, when a global epidemic of cases in non-endemic countries led to the declaration of a Public Health Emergency of International Concern. Cases of mpox in Ireland, a country without previous mpox reports, could reflect extended local transmission or multiple epidemiological introductions.AimTo elucidate the origins and molecular characteristics of MPXV circulating in Ireland between May 2022 and October 2023.MethodsWhole genome sequencing of MPXV from 75% of all Irish mpox cases (182/242) was performed and compared to sequences retrieved from public databases (n = 3,362). Bayesian approaches were used to infer divergence time between sequences from different subclades and evaluate putative importation events from other countries.ResultsOf 242 detected mpox cases, 99% were males (median age: 35 years; range: 15-60). All 182 analysed genomes were assigned to Clade IIb and, presence of 12 distinguishable subclades suggests multiple introductions into Ireland. Estimation of time to divergence of subclades further supports the hypothesis for multiple importation events from numerous countries, indicative of extended and sustained international spread of mpox. Further analysis of sequences revealed that 92% of nucleotide mutations were from cytosine to thymine (or from guanine to adenine), leading to a high number of non-synonymous mutations across subclades; mutations associated with tecovirimat resistance were not observed.ConclusionWe provide insights into the international transmission dynamics supporting multiple introductions of MPXV into Ireland. Such information supported the implementation of evidence-informed public health control measures.

A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

NGS amplification panel for HBV (Hepadnaviridae: <i>Orthohepadnavirus</i>) sequencing

Introduction. Hepatitis B virus (HBV) remains a pressing global public health concern. The clinical course of the disease, particularly its tendency towards chronicity and response to therapy, is significantly influenced by the HBV genotype and specific mutations. There is an imperative need for a straightforward, highly sensitive, and dependable method for whole genome sequencing of HBV.
 Objective. Development and testing of an amplification panel for HBV whole-genome sequencing.
 Materials and methods. We introduce an NGS amplification panel designed for genome sequencing of HBV on the Illumina platform. A panel consisting of 54 primers, divided into 2 pools and amplifying overlapping regions of the HBV genome up to 300 bp in length, was tested on 246 HBV DNA samples.
 Results. The studied samples represented a genotypic diversity of the virus, with a pronounced predominance of the genotype specific to the Moscow region: 216, 27, 2, and 1 sample were identified as genotype D, A, B, and E, respectively. Five samples contained at least one mutation associated with antiviral therapy resistance, and twenty-three samples contained at least one mutation associated with vaccine escape described in the literature.
 Conclusion. The present paper describes the stages of whole-genome sequencing of HBV, provides a laboratory protocol, nucleotide sequences of the primers and an approach to the data analysis. Using a list of clinical samples as example, the reliability of the panel is shown. The HBV panel holds immense potential for utilization in scientific research, epidemiological monitoring, and advancement of personalized medicine approaches.

Current research progress of whole genome sequencing in practical

Gene sequencing, a vital technique nowadays, detects patients diseases and helping with diagnosis. Whole genome sequencing (WGS) is one member of this family, which mainly functions in analysing participants gene sequences in clinical diagnosis for the purpose of indicating therapeutic interventions. This essay compares WGS and other sequencing methods, such as whole exome sequencing (WES), which comprises both positive and negative sides of this sequencing test for evaluation. And point out the help of WGS in certain diseases. This paper serves as a synopsis of some WGS-related themes as well as potential directions in the future for technical advancement.

Mapping crossover events of mouse meiotic recombination by restriction fragment ligation-based Refresh-seq

Single-cell whole-genome sequencing methods have undergone great improvements over the past decade. However, allele dropout, which means the inability to detect both alleles simultaneously in an individual diploid cell, largely restricts the application of these methods particularly for medical applications. Here, we develop a new single-cell whole-genome sequencing method based on third-generation sequencing (TGS) platform named Refresh-seq (restriction fragment ligation-based genome amplification and TGS). It is based on restriction endonuclease cutting and ligation strategy in which two alleles in an individual cell can be cut into equal fragments and tend to be amplified simultaneously. As a new single-cell long-read genome sequencing method, Refresh-seq features much lower allele dropout rate compared with SMOOTH-seq. Furthermore, we apply Refresh-seq to 688 sperm cells and 272 female haploid cells (secondary polar bodies and parthenogenetic oocytes) from F1 hybrid mice. We acquire high-resolution genetic map of mouse meiosis recombination at low sequencing depth and reveal the sexual dimorphism in meiotic crossovers. We also phase the structure variations (deletions and insertions) in sperm cells and female haploid cells with high precision. Refresh-seq shows great performance in screening aneuploid sperm cells and oocytes due to the low allele dropout rate and has great potential for medical applications such as preimplantation genetic diagnosis.

Whole-genome sequencing of some Ukrainian isolates of SARS-COV-2 virus and analysis of its genetic variability

Aim. The aim was to gain experience of the animal health services to detect and properly identify SARS-CoV-2 virus with whole-genome sequencing method and its genetic variability in Ukraine in relation to possible future spread of the virus in animals. Methods. Sixteen SARS-CoV-2 positive samples, not sequenced before, were pro- vided by the Center for Public Health of the Ministry of Health of Ukraine. These samples were obtained from hos- pitalized patients from early October to mid-November of 2021. The viral RNA was isolated from nasopharyngeal swab samples of SARS-CoV-2 qPCR positive (Ct 21-28) patients (males and females) with moderate and severe symptoms who were being hospitalized. The samples were fully anonymized. The Ion Torrent S5 instrument (Ox- ford Nanopore, the USA) was used to sequence the mentioned SARS-CoV-2 isolates, originating from Ukraine. TorrentSuite 5.16.1 was used for data processing and analysis. Nextclade 2.3.0 was used for phylogenetic analysis to locate the 6 sequenced samples on the global phylogenetic tree. It was determined phylogenetic relations be- tween tested 6 sequences and 495 verified sequences of high quality, reported in Ukraine and deposited in the GI- SAID EpiCoVTM database, (https://gisaid.org/) for the period of January 2020 – December 2022. In the compari- son of sequences obtained, the sequence of SARS-CoV-2 virus isolate Wuhan-Hu-1 (GenBank NC_045512.2) was used as a reference sequence, according to which the sequences were aligned. All studies were carried out in the laboratory of the Research Training Center for Animal Disease Diagnostics at the Institute of Veterinary Medicine of the National Academy of Sciences of Ukraine. Results. Among the 16 isolates tested, all were confirmed to con- tain SARS-CoV-2 RNA, of which only six isolates were sequenced with sufficient quality and could be classified, five of them as Delta variants (two belong to lineage AY.126 (B.1.617.2.33), two to AY.122 (B.1.617.2.122), and one to AY.4.2.3 (B.1.617.2.4.2)), and one isolate as an Omicron variant (BA.1.18). Important mutations detected in our isolates were a S:N501Y substitution and S:H69 deletion in the gene of the virus envelope spike protein. Among the examined isolates, the Omicron variant (BA.1.18) was found to exhibit greater genetic variability, with over 60 mutations compared to previous variants. In our investigation, we identified mutations in the sequenced Delta variants too, ranging from 35 mutations in AY.122 (B.1.617.2.122) to 41 mutations in AY.126 (B.1.617.2.33) in the genome compared to the reference Wuhan-Hu-1 (MN908947) variant. Important mutations found regarding infectivity were 1) for the Delta variants: T478K, L452R mutations in the RBD region, and 2) for the Omicron variant: S371L, G339D, S375F, S373P, K417N, N440K, S477N, G446S, E484A, T478K, Q493R, Q498R, G496S, N501Y, and Y505H mutations in the RBD region. Conclusions. The whole-genome sequencing of 6 isolates of SARS-CoV-2 virus was performed, and three sublines of the Delta variant were found: AY.126 (B.1.617.2.33), AY.122 (B.1.617.2.122), AY.4.2.3 (B.1.617.2.4.2) and one subline for the Omicron variant (BA.1.18), all of which were deposited in the international database GISAID as EPI_SET_230516yp. The data obtained in this study add to the existing ones delivered by the Ministry of Health in Ukraine and can be used in laboratories, (including veterinary ones), detecting the SARS-CoV-2 virus in risk animal populations, in order to prevent the spread of the disease to humans and animals, as well as to detect possible mutational changes in the pathogen genome that may affect infectivity and pathogenicity.

Single-cell low-pass whole genome sequencing accurately detects circulating tumor cells for liquid biopsy-based multi-cancer diagnosis

Accurate detection of circulating tumor cells (CTCs) in blood and non-blood body fluids enables generation of deterministic cancer diagnosis and represent a less invasive and safer liquid biopsy approach. Although genomic alternations have been widely used in circulating tumor DNA (ctDNA) analysis, studies on cell-based genomic alternations profiling for CTC detection are rare due to major technical limitations in single-cell whole genome sequencing (WGS) including low throughput, low accuracy and high cost. We report a single-cell low-pass WGS-based protocol (scMet-Seq) for sensitive and accurate CTC detection by combining a metabolic function-associated marker Hexokinase 2 (HK2) and a Tn5 transposome-based WGS method with improved cell fixation strategy. To explore the clinical use, scMet-Seq has been investigated with blood and non-blood body fluids in diagnosing metastatic diseases, including ascites-based diagnosis of malignant ascites (MA) and blood-based diagnosis of metastatic small-cell lung cancer (SCLC). ScMet-Seq shows high diagnostic sensitivity (MA: 79% in >10 cancer types; metastatic SCLC: 90%) and ~100% of diagnostic specificity and positive predictive value, superior to clinical cytology that exhibits diagnostic sensitivity of 52% in MA diagnosis and could not generate blood-based diagnosis. ScMet-Seq represents a liquid biopsy approach for deterministic cancer diagnosis in different types of cancers and body fluids.

Serovar-level identification of bacterial foodborne pathogens from full-length 16S rRNA gene sequencing.

The resolution of variation within species is critical for interpreting and acting on many microbial measurements. In the key foodborne pathogens Salmonella and Escherichia coli, the primary subspecies classification scheme used is serotyping: differentiating variants within these species by surface antigen profiles. Serotype prediction from whole-genome sequencing (WGS) of isolates is now seen as comparable or preferable to traditional laboratory methods where WGS is available. However, laboratory and WGS methods depend on an isolation step that is time-consuming and incompletely represents the sample when multiple strains are present. Community sequencing approaches that skip the isolation step are, therefore, of interest for pathogen surveillance. Here, we evaluated the viability of amplicon sequencing of the full-length 16S rRNA gene for serotyping Salmonella enterica and E. coli. We developed a novel algorithm for serotype prediction, implemented as an R package (Seroplacer), which takes as input full-length 16S rRNA gene sequences and outputs serovar predictions after phylogenetic placement into a reference phylogeny. We achieved over 89% accuracy in predicting Salmonella serotypes on in silico test data and identified key pathogenic serovars of Salmonella and E. coli in isolate and environmental test samples. Although serotype prediction from 16S rRNA gene sequences is not as accurate as serotype prediction from WGS of isolates, the potential to identify dangerous serovars directly from amplicon sequencing of environmental samples is intriguing for pathogen surveillance. The capabilities developed here are also broadly relevant to other applications where intraspecies variation and direct sequencing from environmental samples could be valuable.IMPORTANCEIn order to prevent and stop outbreaks of foodborne pathogens, it is important that we can detect when pathogenic bacteria are present in a food or food-associated site and identify connections between specific pathogenic bacteria present in different samples. In this work, we develop a new computational technology that allows the important foodborne pathogens Escherichia coli and Salmonella enterica to be serotyped (a subspecies level classification) from sequencing of a single-marker gene, and the 16S rRNA gene often used to surveil bacterial communities. Our results suggest current limitations to serotyping from 16S rRNA gene sequencing alone but set the stage for further progress that we consider likely given the rapid advance in the long-read sequencing technologies and genomic databases our work leverages. If this research direction succeeds, it could enable better detection of foodborne pathogens before they reach the public and speed the resolution of foodborne pathogen outbreaks.

Research progress of high precision single base editing technology with a focus on prime editing

Gene editing is a technique in genetic engineering that aims to change specific genes in the genome of a certain organism mainly by modifying specific genes, such as by adding or deleting them. It has a profound meaning in the treatment of human genetic ailments and in the modification of diseased genes in plants and animals, and is also one of the hottest research areas in the life sciences in recent years. This paper focuses on the development of CRISPR/Cas9-based single-base editing technologies after 2019, especially after the advent of lead editing. Researchers in various countries have applied it to rice and mice for experiments to ameliorate the accuracy of single-base editing and decrease off-target effects, and have tested the accuracy and conversion rate of prime editing (PE) by whole-genome sequencing and other methods, and have continued to innovate on this basis to pursue more efficient editing results. This paper summarizes the problems that still need to be solved for PE, so that it can serve as a reference in the future research process and better develop prime editor and gene editing.

Whole genome sequencing of resistance and virulence genes in multi-drug resistant Pseudomonas aeruginosa

BackgroundPseudomonas aeruginosa is an opportunistic bacterium that causes serious hospital-acquired infections. To assess the risk of clinically isolated P. aeruginosa to human health, we analyzed the resistance and virulence mechanisms of a collection of clinical isolates. MethodsThis was a retrospective study in which P. aeruginosa isolates collected from January 1, 2018 to August 31, 2019 were analyzed using phenotypic and whole-genome sequencing (WGS) methods. The analysis included 48 clinical samples. Median patient age was 54.0 (29.5) years, and 58.3% of patients were women. Data from the microbiology laboratory database were reviewed to identify P. aeruginosa isolates. All unique isolates available for further testing were included, and related clinical data were collected. Infections were defined as hospital acquired if the index culture was obtained at least 48 h after hospitalization. ResultsHigh-risk P. aeruginosa clones, including sequence types (STs) ST235 and ST111, were identified, in addition to 12 new STs. The isolates showed varying degrees of biofilm formation ability when evaluated at room temperature, along with reduced metabolic activity, as measured by metabolic staining, suggesting their ability to evade antimicrobial therapy. Most isolates (77.1%) were multidrug resistant (MDR), with the highest resistance and susceptibility rates to beta-lactams and colistimethate sodium, respectively. ConclusionsThe MDR phenotypes of the examined isolates can be explained by the high prevalence of efflux-mediated resistance- and hydrolytic enzyme-encoding genes. These isolates had high cytotoxic potential, as indicated by the detection of toxin production-related genes.

Search for differentially methylated regions in ancient and modern genomes

Currently, active research is focused on investigating the mechanisms that regulate the development of various pathologies and their evolutionary dynamics. Epigenetic mechanisms, such as DNA methylation, play a significant role in evolutionary processes, as their changes have a faster impact on the phenotype compared to mutagenesis. In this study, we attempted to develop an algorithm for identifying differentially methylated regions associated with metabolic syndrome, which have undergone methylation changes in humans during the transition from a hunter­gatherer to a sedentary lifestyle. The application of existing whole­genome bisulfite sequencing methods is limited for ancient samples due to their low quality and fragmentation, and the approach to obtaining DNA methylation profiles differs significantly between ancient hunter­gatherer samples and modern tissues. In this study, we validated DamMet, an algorithm for reconstructing ancient methylomes. Application of DamMet to Neanderthal and Denisovan genomes showed a moderate level of correlation with previously published methylation profiles and demonstrated an underestimation of methylation levels in the reconstructed profiles by an average of 15–20 %. Additionally, we developed a new Python­based algorithm that allows for the comparison of methylomes in ancient and modern samples, despite the absence of methylation profiles in modern bone tissue within the context of obesity. This analysis involves a two­step data processing approach, where the first step involves the identification and filtration of tissue­specific methylation regions, and the second step focuses on the direct search for differentially methylated regions in specific areas associated with the researcher’s target condition. By applying this algorithm to test data, we identified 38 differentially methylated regions associated with obesity, the majority of which were located in promoter regions. The pipeline demonstrated sufficient efficiency in detecting these regions. These results confirm the feasibility of reconstructing DNA methylation profiles in ancient samples and comparing them with modern methylomes. Furthermore, possibilities for further methodological development and the implementation of a new step for studying differentially methylated positions associated with evolutionary processes are discussed.

A Review on Microbiological Source Attribution Methods of Human Salmonellosis: From Subtyping to Whole-Genome Sequencing.

Salmonella is one of the main causes of human foodborne illness. It is endemic worldwide, with different animals and animal-based food products as reservoirs and vehicles of infection. Identifying animal reservoirs and potential transmission pathways of Salmonella is essential for prevention and control. There are many approaches for source attribution, each using different statistical models and data streams. Some aim to identify the animal reservoir, while others aim to determine the point at which exposure occurred. With the advance of whole-genome sequencing (WGS) technologies, new source attribution models will greatly benefit from the discriminating power gained with WGS. This review discusses some key source attribution methods and their mathematical and statistical tools. We also highlight recent studies utilizing WGS for source attribution and discuss open questions and challenges in developing new WGS methods. We aim to provide a better understanding of the current state of these methodologies with application to Salmonella and other foodborne pathogens that are common sources of illness in the poultry and human sectors.

Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance.

The COVID-19 pandemic has necessitated the rapid development and implementation of whole-genome sequencing (WGS) and bioinformatic methods for managing the pandemic. However, variability in methods and capabilities between laboratories has posed challenges in ensuring data accuracy. A national working group comprising 18 laboratory scientists and bioinformaticians from Australia and New Zealand was formed to improve data concordance across public health laboratories (PHLs). One effort, presented in this study, sought to understand the impact of the methodology on consensus genome concordance and interpretation. SARS-CoV-2 WGS proficiency testing programme (PTP) data were retrospectively obtained from the 2021 Royal College of Pathologists of Australasia Quality Assurance Programmes (RCPAQAP), which included 11 participating Australian laboratories. The submitted consensus genomes and reads from eight contrived specimens were investigated, focusing on discordant sequence data and findings were presented to the working group to inform best practices. Despite using a variety of laboratory and bioinformatic methods for SARS-CoV-2 WGS, participants largely produced concordant genomes. Two participants returned five discordant sites in a high-Cτ replicate, which could be resolved with reasonable bioinformatic quality thresholds. We noted ten discrepancies in genome assessment that arose from nucleotide heterogeneity at three different sites in three cell-culture-derived control specimens. While these sites were ultimately accurate after considering the participants' bioinformatic parameters, it presented an interesting challenge for developing standards to account for intrahost single nucleotide variation (iSNV). Observed differences had little to no impact on key surveillance metrics, lineage assignment and phylogenetic clustering, while genome coverage <90 % affected both. We recommend PHLs bioinformatically generate two consensus genomes with and without ambiguity thresholds for quality control and downstream analysis, respectively, and adhere to a minimum 90 % genome coverage threshold for inclusion in surveillance interpretations. We also suggest additional PTP assessment criteria, including primer efficiency, detection of iSNVs and minimum genome coverage of 90 %. This study underscores the importance of multidisciplinary national working groups in informing guidelines in real time for bioinformatic quality acceptance criteria. It demonstrates the potential for enhancing public health responses through improved data concordance and quality control in SARS-CoV-2 genomic analysis during pandemic surveillance.

EPCO-46. CHARACTERIZING ATYPICAL TERATOID RHABDOID TUMOR SUBTYPE ENHANCER INTERACTION LANDSCAPE

Abstract Atypical Teratoid Rhabdoid Tumor (ATRT) is one of the most aggressive pediatric tumors of the central nervous system (CNS). The majority of ATRTs are characterized by the recurrent bi-allelic loss of the SWI/SNF nucleosome remodeling complex core component gene, SMARCB1, or SMARCA4 in rare cases. Despite its bland genome, three ATRT subgroups “SHH”, “TYR”, and “MYC” were discovered with unique molecular profiles. Also, distinct activating histone mark H3K27ac profiles between molecular subgroups were identified, suggesting subgroup-specific regulatory networks through enhancers. However, comprehensive cis-regulatory network descriptions in ATRT are lacking. Here, we aim to elucidate ATRT subgroup pathogenesis through its dysregulated enhancer-promoter interactions. We have created a regulatory mutational profile of the three ATRT subgroups using whole-genome sequencing method of 60 ATRT tumors and matching genome-wide H3K27ac profiles on 16 tumors. To investigate mutation burden on cis-regulations, we have employed high-throughput Hi-C chromatin immunoprecipitation (HiChIP) technique to directly map enhancer sequences involved in DNA-looping with high specificity and sensitivity. Recent findings from our lab suggest that activating interaction PDGFRB confer to therapeutics venerability in the MYC subgroup. Here, our preliminary analysis shows a hub of interactions near PDGFRB reveals an extensive interaction hub with long-range enhancer interactions extending as far as 1500Kb, suggesting a much more complicated regulatory picture than previously expected. Interestingly, we have identified long-range DNA interactions between PDGFRB and H3K27ac marks upstream of its gene body and overstretching into other gene elements. This result suggests a potential alternative activation mechanism for MYC cell lines. Our next steps are to perform performing additional HiChIP assays on other ATRT cell lines from other subgroups to capture the full range of ATRT’s heterogeneity. Comprehensive mapping of the non-coding genome and enhancer regulatory network would help identify critical pathogenic elements underlying ATRT biology and provide insights into therapeutic targets.

Novel TFG mutation causes autosomal-dominant spastic paraplegia and defects in autophagy

BackgroundMutations in the tropomyosin receptor kinase fused (TFG) gene are associated with various neurological disorders, including autosomal recessive hereditary spastic paraplegia (HSP), autosomal dominant hereditary motor and sensory neuropathy with...

Loss of LRP1B expression drives acquired chemo and radio-resistance in HPV-positive head and neck cancer

ObjectivesAlthough human papillomavirus positive (HPV+) oropharyngeal squamous cell carcinoma (OPSCC) patients typically experience excellent survival, 15–20 % of patients recur after treatment with chemotherapy and radiation. Therefore, there is a need for biomarkers of treatment failure to guide treatment intensity. Materials and methodsWhole genome sequencing was carried out on HPV+OPSCC patients who were primarily treated with concurrent chemotherapy (cisplatin) and radiation. We then explored whether the loss of LRP1Bwas sufficient to drive an aggressive phenotype, and promote a resistance to cisplatin and radiation therapy both in vitro using HPV+ cell lines (93VU147T, UMSCC47, UWO37 and UWO23) and in vivo. ResultsThrough integrative genomic analysis of three HPV+OPSCC tumour datasets, we identified that deletion of LRP1B was enriched in samples that recurred following chemo-radiation. Knockdown using siRNA in four HPV+ cell lines (UWO23, UWO37, UMSCC47 and 93VU147T) resulted in increased proliferation of all cases. CRISPR/Cas9 deletion of LRP1B in the same cell line panel demonstrated increased proliferation, clonogenic growth and migration, as well as resistance to both cisplatin and radiation in LRP1B deleted cells compared to their respective non-targeting control cells. Cell line derived xenograft studies indicated that the LRP1B knockout tumours were more resistant to cisplatin and radiation therapy compared to their controls invivo. ConclusionTaken together, our work implicates LRP1B deletion as a potential biomarker for identifying treatment resistant HPV+ OPSCC cases.

Status and physiological significance of circulating adiponectin in the very old and centenarians: an observational study.

High levels of circulating adiponectin are associated with increased insulin sensitivity, low prevalence of diabetes, and low body mass index (BMI); however, high levels of circulating adiponectin are also associated with increased mortality in the 60-70 age group. In this study, we aimed to clarify factors associated with circulating high-molecular-weight (cHMW) adiponectin levels and their association with mortality in the very old (85-89 years of age) and centenarians. The study included 812 (women: 84.4%) for centenarians and 1498 (women: 51.7%) for the very old. The genomic DNA sequence data were obtained by whole-genome sequencing or DNA microarray-imputation methods. Least Absolute Shrinkage and Selection Operator (LASSO) and multivariate regression analyses were used to evaluate cHMW adiponectin characteristics and associated factors. All-cause mortality was analyzed in three quantile groups of cHMW adiponectin levels using Cox regression. The cHMW adiponectin levels were increased significantly beyond 100 years of age, were negatively associated with diabetes prevalence, and were associated with SNVs in CDH13 (p=2.21 × 10-22) and ADIPOQ (p=5.72 × 10-7). Multivariate regression analysis revealed that genetic variants, BMI, and high-density lipoprotein cholesterol (HDLC) were the main factors associated with cHMW adiponectin levels in the very old, whereas the BMI showed no association in centenarians. The hazard ratios for all-cause mortality in the intermediate and high cHMW adiponectin groups in very old men were significantly higher rather than those for all-cause mortality in the low-level cHMW adiponectin group, even after adjustment with BMI. In contrast, the hazard ratios for all-cause mortality were significantly higher for high cHMW adiponectin groups in very old women, but were not significant after adjustment with BMI. cHMW adiponectin levels increased with age until centenarians, and the contribution of known major factors associated with cHMW adiponectin levels, including BMI and HDLC, varies with age, suggesting that its physiological significance also varies with age in the oldest old. This study was supported by grants from the Ministry of Health, Welfare, and Labour for the Scientific Research Projects for Longevity; a Grant-in-Aid for Scientific Research (No 21590775, 24590898, 15KT0009, 18H03055, 20K20409, 20K07792, 23H03337) from the Japan Society for the Promotion of Science; Keio University Global Research Institute (KGRI), Kanagawa Institute of Industrial Science and Technology (KISTEC), Japan Science and Technology Agency (JST) Research Complex Program "Tonomachi Research Complex" Wellbeing Research Campus: Creating new values through technological and social innovation (JP15667051), the Program for an Integrated Database of Clinical and Genomic Information from the Japan Agency for Medical Research and Development (No. 16kk0205009h001, 17jm0210051h0001, 19dk0207045h0001); the medical-welfare-food-agriculture collaborative consortium project from the Japan Ministry of Agriculture, Forestry, and Fisheries; and the Biobank Japan Program from the Ministry of Education, Culture, Sports, and Technology.

Establishment and Evaluation of a Multiplex Pcr amplification-based Sequencing Method for Respiratory Virus Type A

Aim: This study aimed to establish a simple whole-genome sequencing method for respiratory virus type A (RSVA) used in genomic epidemiological studies. Methods: Multiple primers were designed to amplify overlapping amplicons specific to RSVA. The amplicons were sequenced using Illumina and Nanopore sequencing platforms and the sequencing performances of the two platforms were also compared. Results: The study shows that the developed method could recover almost full genomes of RSVA when Ct values were less than 33 for clinical samples. Conclusion: In this study, a whole-genome sequencing method was developed for RSVA based on multiplex PCR.