Sequencing Strategy Research Articles

Diallel panel reveals a significant impact of low-frequency genetic variants on gene expression variation in yeast.

Unraveling the genetic sources of gene expression variation is essential to better understand the origins of phenotypic diversity in natural populations. Genome-wide association studies identified thousands of variants involved in gene expression variation, however, variants detected only explain part of the heritability. In fact, variants such as low-frequency and structural variants (SVs) are poorly captured in association studies. To assess the impact of these variants on gene expression variation, we explored a half-diallel panel composed of 323 hybrids originated from pairwise crosses of 26 natural Saccharomyces cerevisiae isolates. Using short- and long-read sequencing strategies, we established an exhaustive catalog of single nucleotide polymorphisms (SNPs) and SVs for this panel. Combining this dataset with the transcriptomes of all hybrids, we comprehensively mapped SNPs and SVs associated with gene expression variation. While SVs impact gene expression variation, SNPs exhibit a higher effect size with an overrepresentation of low-frequency variants compared to common ones. These results reinforce the importance of dissecting the heritability of complex traits with a comprehensive catalog of genetic variants at the population level.

Do we still need Illumina sequencing data? Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and the Rapid v14 library prep kit for Gram negative bacteria whole genome assemblies.

The best whole genome assemblies are currently built from a combination of highly accurate short-read sequencing data and long-read sequencing data that can bridge repetitive and problematic regions. Oxford Nanopore Technologies (ONT) produce long-read sequencing platforms and they are continually improving their technology to obtain higher quality read data that is approaching the quality obtained from short-read platforms such as Illumina. As these innovations continue, we evaluated how much ONT read coverage produced by the Rapid Barcoding Kit v14 (SQK-RBK114) is necessary to generate high-quality hybrid and long-read-only genome assemblies for a panel of carbapenemase-producing Enterobacterales bacterial isolates. We found that 30× long-read coverage is sufficient if Illumina data are available, and that more (at least 100× long-read coverage is recommended for long-read-only assemblies. Illumina polishing is still improving single nucleotide variants (SNVs) and INDELs in long-read-only assemblies. We also examined if antimicrobial resistance genes could be accurately identified in long-read-only data, and found that Flye assemblies regardless of ONT coverage detected>96% of resistance genes at 100% identity and length. Overall, the Rapid Barcoding Kit v14 and long-read-only assemblies can be an optimal sequencing strategy (i.e., plasmid characterization and AMR detection) but finer-scale analyses (i.e., SNV) still benefit from short-read data.

A Deep Sequencing Strategy for Investigation of Virus Variants within African Swine Fever Virus-Infected Pigs.

African swine fever virus (ASFV) is the causative agent of African swine fever, an economically important disease of pigs, often with a high case fatality rate. ASFV has demonstrated low genetic diversity among isolates collected within Eurasia. To explore the influence of viral variants on clinical outcomes and infection dynamics in pigs experimentally infected with ASFV, we have designed a deep sequencing strategy. The variant analysis revealed unique SNPs at <10% frequency in several infected pigs as well as some SNPs that were found in more than one pig. In addition, a deletion of 10,487 bp (resulting in the complete loss of 21 genes) was present at a nearly 100% frequency in the ASFV DNA from one pig at position 6362-16849. This deletion was also found to be present at low levels in the virus inoculum and in two other infected pigs. The current methodology can be used for the currently circulating Eurasian ASFVs and also adapted to other ASFV strains and genotypes. Comprehensive deep sequencing is critical for following ASFV molecular evolution, especially for the identification of modifications that affect virus virulence.

Genomic Surveillance of SARS-CoV-2: Data Analysis and Assessment of Tunisian Strategy from January 2021 to February 2022.

Due to the emergence of the SARS-CoV-2 B.1.1.7 (Alpha) variant in the UK in 2020 and its risk of increased transmission, the Ministry of Health in Tunisia implemented a sequencing surveillance strategy for SARS-CoV-2. The aim of this study was to analyze SARS-CoV-2 genomic surveillance data in Tunisia (January 2021-February 2022) and to assess the implementation of the sequencing strategy for SARS-CoV-2 in accordance with national recommendations and the guidance for SARS-CoV-2 genomic surveillance for public health goals. A descriptive study of all sequenced RT-PCR samples sequenced (January 2021-February2022). An internal audit was also done to assess the compliance against standards covering national recommendations and the Guidance for SARS-CoV-2 genomic surveillance for public health goals. A total of 12 simple or composite requirements related to the following areas were included in the audit standards: sampling (one requirements); data collection/analysis (six requirements); partnership (one requirement); and ethical considerations (one requirement). A total of 4819 samples were sent to laboratories and 4278 samples were sequenced. A total of 3648 samples were classified. Positive variants of concern (VOC) samples were 80.92%, differentiated as follows: Alpha, 40.24%; Beta, 0.24%; Gamma, 0.03%; Delta, 45.26%; and Omicron, 14.19%. Three principal phases of VOCs per ISO-week were shown: Alpha 3/2021-25/2021; Delta 26/2021-2/2022; and Omicron 3/2022-6/2022. Levels of compliance were identified; from a total of 12 requirements, 7 were considered as "not met", 4 as "partially met", and 1 as "fully met" but including not totally achieved objectives. In conclusion, the internal audit of the national SARS-CoV-2 sequencing strategy revealed an overall "not met" level of compliance. The results offered a trigger to collaborate with all stakeholders to develop a surveillance strategy for early detection and response to outbreaks caused by VOCs.

Diagnostic strategy of metagenomic next-generation sequencing for gram negative bacteria in respiratory infections

ObjectiveThis study aims to identify the most effective diagnostic method for distinguishing pathogenic and non-pathogenic Gram-negative bacteria (GNB) in suspected pneumonia cases using metagenomic next-generation sequencing (mNGS) on bronchoalveolar lavage fluid (BALF) samples.MethodsThe effectiveness of mNGS was assessed on BALF samples collected from 583 patients, and the results were compared with those from microbiological culture and final clinical diagnosis. Three interpretational approaches were evaluated for diagnostic accuracy.ResultsmNGS outperformed culture significantly. Among the interpretational approaches, Clinical Interpretation (CI) demonstrated the best diagnostic performance with a sensitivity of 87.3%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 98.3%. CI’s specificity was significantly higher than Simple Interpretation (SI) at 37.9%. Additionally, CI excluded some microorganisms identified as putative pathogens by SI, including Haemophilus parainfluenzae, Haemophilus parahaemolyticus, and Klebsiella aerogenes.ConclusionProper interpretation of mNGS data is crucial for accurately diagnosing respiratory infections caused by GNB. CI is recommended for this purpose.

RNA polymerase II pausing is essential during spermatogenesis for appropriate gene expression and completion of meiosis

Male germ cell development requires precise regulation of gene activity in a cell-type and stage-specific manner, with perturbations in gene expression during spermatogenesis associated with infertility. Here, we use steady-state, nascent and single-cell RNA sequencing strategies to comprehensively characterize gene expression across male germ cell populations, to dissect the mechanisms of gene control and provide new insights towards therapy. We discover a requirement for pausing of RNA Polymerase II (Pol II) at the earliest stages of sperm differentiation to establish the landscape of gene activity across development. Accordingly, genetic knockout of the Pol II pause-inducing factor NELF in immature germ cells blocks differentiation to spermatids. Further, we uncover unanticipated roles for Pol II pausing in the regulation of meiosis during spermatogenesis, with the presence of paused Pol II associated with double-strand break (DSB) formation, and disruption of meiotic gene expression and DSB repair in germ cells lacking NELF.

Domestication signatures in the non-conventional yeast Lachancea cidri.

Evaluating domestication signatures beyond model organisms is essential for a thorough understanding of the genotype-phenotype relationship in wild and human-related environments. Structural variations (SVs) can significantly impact phenotypes playing an important role in the physiological adaptation of species to different niches, including during domestication. A detailed characterization of the fitness consequences of these genomic rearrangements, however, is still limited in non-model systems, largely due to the paucity of direct comparisons between domesticated and wild isolates. Here, we used a combination of sequencing strategies to explore major genomic rearrangements in a Lachancea cidri yeast strain isolated from cider (CBS2950) and compared them to those in eight wild isolates from primary forests. Genomic analysis revealed dozens of SVs, including a large reciprocal translocation (~16 kb and 500 kb) present in the cider strain, but absent from all wild strains. Interestingly, the number of SVs was higher relative to single-nucleotide polymorphisms in the cider strain, suggesting a significant role in the strain's phenotypic variation. The set of SVs identified directly impacts dozens of genes and likely underpins the greater fermentation performance in the L. cidri CBS2950. In addition, the large reciprocal translocation affects a proline permease (PUT4) regulatory region, resulting in higher PUT4 transcript levels, which agrees with higher ethanol tolerance, improved cell growth when using proline, and higher amino acid consumption during fermentation. These results suggest that SVs are responsible for the rapid physiological adaptation of yeast to a human-related environment and demonstrate the key contribution of SVs in adaptive fermentative traits in non-model species.IMPORTANCEThe exploration of domestication signatures associated with human-related environments has predominantly focused on studies conducted on model organisms, such as Saccharomyces cerevisiae, overlooking the potential for comparisons across other non-Saccharomyces species. In our research, employing a combination of long- and short-read data, we found domestication signatures in Lachancea cidri, a non-model species recently isolated from fermentative environments in cider in France. The significance of our study lies in the identification of large array of major genomic rearrangements in a cider strain compared to wild isolates, which underly several fermentative traits. These domestication signatures result from structural variants, which are likely responsible for the phenotypic differences between strains, providing a rapid path of adaptation to human-related environments.

The pan-microbiome profiling system Taxa4Meta identifies clinical dysbiotic features and classifies diarrheal disease.

Targeted metagenomic sequencing is an emerging strategy to survey disease-specific microbiome biomarkers for clinical diagnosis and prognosis. However, this approach often yields inconsistent or conflicting results owing to inadequate study power and sequencing bias. We introduce Taxa4Meta, a bioinformatics pipeline explicitly designed to compensate for technical and demographic bias. We designed and validated Taxa4Meta for accurate taxonomic profiling of 16S rRNA amplicon data acquired from different sequencing strategies. Taxa4Meta offers significant potential in identifying clinical dysbiotic features that can reliably predict human disease, validated comprehensively via reanalysis of individual patient 16S data sets. We leveraged the power of Taxa4Meta's pan-microbiome profiling to generate 16S-based classifiers that exhibited excellent utility for stratification of diarrheal patients with Clostridioides difficile infection, irritable bowel syndrome, or inflammatory bowel diseases, which represent common misdiagnoses and pose significant challenges for clinical management. We believe that Taxa4Meta represents a new "best practices" approach to individual microbiome surveys that can be used to define gut dysbiosis at a population-scale level.

Abstract A001: Preoperative treatment with mFOLFIRINOX +/- isotoxic high-dose stereotactic body radiation therapy (iHD-SBRT) for borderline resectable pancreatic adenocarcinoma (the STEREOPAC trial): A randomized comparative multicenter phase II trial

Abstract Background: Surgical resection is the only potentially curative treatment for patients with pancreatic ductal adenocarcinoma (PDAC). Neoadjuvant therapy is a rapidly emerging concept that needs to be explored and validated in terms of therapeutic options and survival benefit. In this setting, preoperative modified FOLFIRINOX (mFFX) seems to be feasible and can be followed by radiation therapy with promising benefit for patients in terms of R0 resection, local control and survival. However, the best therapeutic sequence is not yet known and the role of adding high-dose SBRT to chemotherapy in the neoadjuvant strategy requires assessment in randomized trials. The aim of the study is to evaluate the impact and efficacy of adding isotoxic high-dose SBRT (iHD-SBRT) to neoadjuvant mFFX in patients with borderline resectable PDAC. Methods: This is a randomized comparative multicentre phase II trial, planning to enrol patients (n= 256) diagnosed with a borderline resectable biopsy-confirmed PDAC. Patients will receive 4 cycles of mFFX (or 6 doses of gemcitabine/nab-paclitaxel [Gem/Nab-P] in case of intolerance). After full disease restaging, non-progressive patients will be randomized for receiving either 4 additional mFFX cycles (or 6 doses of Gem/Nab-P) (Arm A), or 2 mFFX cycles (or 3 doses of Gem/Nab-P) + iHD-SBRT (35 to 55Gy in 5 fractions) + 2 mFFX cycles (or 3 doses of Gem/Nab-P) (Arm B). Then curative surgery will be performed followed by adjuvant chemotherapy according to patient’s condition. The co-primary endpoints are R0 resection and disease-free survival after the complete sequence strategy. The secondary endpoints include resection rate, overall survival, locoregional failure/distant metastasis free interval, pathologic complete response, toxicity, postoperative complications and quality of life assessment. Discussion: This trial will help define the best neoadjuvant treatment sequence for borderline resectable PDAC and aims to evaluate if a total neoadjuvant treatment integrating iHD-SBRT improves the patients’ oncological outcomes. Trial registration: The study was registered at ClinicalTrails.gov (NCT05083247) on October 19th, 2021, and in the Clinical Trials Information System (CTIS) EU CT database (2022-501181-22-01) on July 2022. The first patient was included on March 24, 2023. Citation Format: Christelle Bouchart, Julie Navez, Ivan Borbath, Karen Geboes, Timon Vandamme, Jean Closset, Luigi Moretti, Pieter Demetter, Marianne Paesmans, Jean-Luc Van Laethem. Preoperative treatment with mFOLFIRINOX +/- isotoxic high-dose stereotactic body radiation therapy (iHD-SBRT) for borderline resectable pancreatic adenocarcinoma (the STEREOPAC trial): A randomized comparative multicenter phase II trial [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A001.

An Analysis of the Gut Microbiota and Related Metabolites following PCSK9 Inhibition in Statin-Treated Patients with Elevated Levels of Lipoprotein(a).

Atherosclerotic cardiovascular disease (ASCVD) is a leading cause of global mortality, often associated with high blood levels of LDL cholesterol (LDL-c). Medications like statins and PCSK9 inhibitors, are used to manage LDL-c levels and reduce ASCVD risk. Recent findings connect the gut microbiota and its metabolites to ASCVD development. We showed that statins modulate the gut microbiota including the production of microbial metabolites involved in the regulation of cholesterol metabolism such as short chain fatty acids (SCFAs) and bile acids (BAs). Whether this pleiotropic effect of statins is associated with their antimicrobial properties or it is secondary to the modulation of cholesterol metabolism in the host is unknown. In this observational study, we evaluated whether alirocumab, a PCSK9 inhibitor administered subcutaneously, alters the stool-associated microbiota and the profiles of SCFAs and BAs. We used stool and plasma collected from patients enrolled in a single-sequence study using alirocumab. Microbial DNA was extracted from stool, and the bacterial component of the gut microbiota profiled following an amplicon sequencing strategy targeting the V3-V4 region of the 16S rRNA gene. Bile acids and SCFAs were profiled and quantified in stool and plasma using mass spectrometry. Treatment with alirocumab did not alter bacterial alpha (Shannon index, p = 0.74) or beta diversity (PERMANOVA, p = 0.89) in feces. Similarly, circulating levels of SCFAs (mean difference (95% confidence interval (CI)), 8.12 [-7.15-23.36] µM, p = 0.25) and BAs (mean difference (95% CI), 0.04 [-0.11-0.19] log10(nmol mg-1 feces), p = 0.56) were equivalent regardless of PCSK9 inhibition. Alirocumab therapy was associated with increased concentration of BAs in feces (mean difference (95% CI), 0.20 [0.05-0.34] log10(nmol mg-1 feces), p = 0.01). In statin-treated patients, the use of alirocumab to inhibit PCSK9 leads to elevated levels of fecal BAs without altering the bacterial population of the gut microbiota. The association of alirocumab with increased fecal BA concentration suggests an additional mechanism for the cholesterol-lowering effect of PCSK9 inhibition.

A dynamic multi-objective evolutionary algorithm based on genetic engineering and improved particle swarm prediction strategy

Evolutionary and particle swarm optimisation (PSO) algorithms are widely used to solve various optimisation problems. However, both methods have limitations when applied to dynamic multi-objective optimisation problems. Therefore, we propose a dynamic multi-objective evolutionary algorithm based on genetic engineering and improved particle swarm prediction. Genetic engineering encompasses gene sequencing and editing, whereas improved particle swarm prediction involves enhancing information utilisation and dynamic performance. The improved PSO can be better combined with dynamic multi-objective optimization problems (DMOPs) for predicting changing populations, which we call improved particle swarm prediction. Gene sequencing clarifies the functions of different genes during the evolution process, and gene editing enables the algorithm to effectively avoid local optima. Expanding on this foundation, we redefined the notions of particle and global bests in the PSO strategy to improve the information utilisation. Additionally, we innovatively introduced the concept of acceleration, which greatly enhances the dynamic performance of the PSO. Finally, we conducted two experiments to verify the accuracy of our gene sequencing strategy and the performance of our optimisation algorithm. The results indicate that gene sequencing can effectively classify genes, and the developed optimisation algorithm significantly outperforms similar, state-of-the-art, and classical algorithms in terms of distribution and dynamics on 25 test problems.

Portable Nanopore sequencing solution for next-generation HIV drug resistance testing

BackgroundTackling HIV drug resistance is one of major challenges for ending AIDS epidemic, but the elevated expense of cutting-edge genomics hampers the advancement of HIV genotype testing for clinical care. MethodsWe developed a HIV genotype testing pipeline that centers on a cost-efficient portable Nanopore sequencer. Accuracy verification was conducted through comparison with parallel data obtained via fixed-site Pacbio sequencing. Our complete pol-gene sequencing strategy coupled with portable high-throughput sequencing was applied to identify drug resistance mutations across 58 samples sourced from the ART-treated Los Angeles General Medical Center Rand Schrader Clinic (LARSC) cohort (7 samples from 7 individuals) and the ART-naïve Center for HIV/AIDS Vaccine Immunology (CHAVI) cohort (51 samples from 38 individuals). ResultsA total of 472 HIV consensus sequences, each tagged with a unique molecular identifier, were produced from over 1.4 million bases acquired through portable Nanopore sequencing, which matched those obtained independently via Pacbio sequencing. With this desirable accuracy, we first documented the linkage of multidrug cross-resistance mutations across Integrase Strand Transfer inhibitors (INSTIs) and Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs) from an individual failing a second-generation INSTI regimen. By producing more than 500 full-length HIV pol gene sequences in a single portable sequencing run, we detected Protease Inhibitor (PI), Nucleoside Reverse Transcriptase Inhibitor (NRTI), NNRTI and INSTI resistance mutations. All drug resistance mutations identified through portable sequencing were cross-validated using fixed-site Pacbio sequencing. ConclusionsOur accurate and affordable HIV drug resistance testing solution is adaptable for both individual patient care and large-scale surveillance initiatives.

Single agent vs combination immunotherapy in advanced melanoma: a review of the evidence.

The aim of this review is to outline the current landscape of advanced melanoma treatment options, provide insights on selecting combination therapies within different clinical scenarios, capture clinical relevance of anti-programmed cell death protein 1 (PD-1) monotherapy, and explore the unmet needs with immune check-point inhibitors (ICI) in advanced melanoma. ICI based treatment consisted of single agent ICI or dual combination ICI-ICI is the standard of care of front-line treatment of metastatic or unresectable melanoma. PD-1 inhibitors (Pembrolizumab and Nivolumab) improved progression free survival (PFS) and overall survival (OS) compared to chemotherapy and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) inhibitors (Ipilimumab and Tremelimumab). The dual ICI combination (Nivolumab and Ipilimumab) provided profound and durable responses better than monotherapy, and the longest overall survival ever achieved in advanced disease, including in patients with murine sarcoma viral oncogene homolog B (BRAF)-mutated disease, but at the cost of a high risk of severe toxicity. The new dual blockage of LAG-3 and PD-1 (Nivolumab-Relatlimab) emerges as a valid option with promising efficacy outcomes and a favourable toxicity profile. Mature survival data is still needed to capture the real benefit. These new plethora of options pose new challenges not only for optimal treatment sequencing strategies but especially for management of adverse effects, endorsing the need to integrate a holistic and personalized approach for patient care.

Metastatic Prostate Cancer: The Optimal Sequencing and Bone Health Management Strategies

Summary Remarkable progress has been made in the treatment of metastatic prostate cancer in the last decade. Prior to initiation of therapy, risk stratification is of paramount importance. Androgen deprivation therapy along with newer systemic therapies are the mainstay of treatment in the metastatic setting. Local therapy and bone directed therapy are important considerations. In this review we discuss the appropriate sequencing strategies in metastatic prostate cancer.

Business development strategy of coffee processing in MSMEs scale using BMC approach and SWOT-AHP integration

The coffee agro-industry is a sector that is currently growing rapidly in Indonesia. MSMEs Loca Nusa is one of the coffee producers in Malang City which has the potential to be developed. They sell robusta coffee at low prices and delivered from Dampit, Malang which is known to have the best quality. However, Loca Nusa still experience problems in managing their business. The purpose of this study is to identify internal and external factors, formulate and sequence strategies, and form a new development BMC. This study used SWOT (Strength, Weakness, Opportunity, Threat), the AHP (Analytical Hierarchy Process) and BMC (Business Model Canvas) approach. The results showed that the position of MSMEs Loca Nusa was in quadrant V (hold and maintain) with product development and market penetration strategies. The result of the AHP ranking showed market as the highest criteria (0.510), the highest goal is that MSMEs can expand the market to increase production figures (0,566), and the highest alternative is expanding the market to coffee shops in Malang by selling roasted bean products with various variations (0,612). The addition of strategy to the upgraded BMC is on key partner elements, value propositions, and channels.

Effect of Cutting Conditions on Surface Integrity when Robotic Drilling of Aluminum 6082-GFRP Stacks

Six-axis robots are increasingly employed in manufacturing due to their excellent volume on cost ratio and extensive reach, facilitating the machining of large components, including those made of composites. However, this enhanced volume on cost ratio often compromises rigidity. This study investigates the effect of cutting conditions on surface integrity and robotic drilling forces when machining Aluminum 6082-GFRP stacks using 6 mm solid carbide drills and a 6-axis Sta¨ubli TX200 robot. Two distinct drill geometries are assessed in various drilling sequences. Feed rate emerges as a pivotal parameter affecting drilling forces and hole quality. Comprehensive testing encompasses a range of feed rates and drilling strategies in both individual materials and stack sequences. The analysis includes cutting forces and hole quality, considering surface integrity and standard quality indicators such as delamination, arithmetic roughness, and burr height. The research seeks to propose optimal cutting conditions, specifically feed rate and cutting speed, essential for advanced industrial applications. These conditions ensure hole quality and surface integrity, which is particularly important for riveting processes and ensuring effective sealing.

The Intra-Host Evolution of SARS-CoV-2 After Neutralizing Antibody Therapy, Revealed by Nanopore Sequencing

Objective: In the context of two Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreaks involving local transmission and an international flight, we used meta-transcriptome and multi-amplicon sequencing to successfully acquire the complete viral genome sequences from clinical samples with varying viral loads. Methods: To enhance viral transcript presence, we used a primer pool for reverse transcription and sequenced the samples with nanopore sequencing, and successfully acquired the entire genomic sequence of the virus within less than 4 hours. In a substantial sample size of approximately 800 clinical specimens, we thoroughly examined and compared different sequencing methods. Results: Meta-transcriptome sequencing was effective for samples with viral reverse transcription polymerase chain reaction (RT-PCR) threshold cycle (Ct) values below 22, whereas multi-amplicon sequencing was effective across a wide Ct range. Additionally, enriched nanopore sequencing was valuable in capturing the complete genome sequence when rapid results are required. Conclusion: Through monitoring the viral quasi-species in individual patients, we observed ongoing viral evolution during neutralizing antibody therapy and found evidence that vaccine administration may affect the development of viral quasi-species. Overall, our findings highlight the potential of this viral sequencing strategy for both outbreak control and patient treatment.

Detection and full genomic sequencing of rare hepatitis E virus genotype 4d in Italian wastewater, undetected by clinical surveillance

Hepatitis E is a liver disease caused by the hepatitis E virus (HEV), primarily transmitted through contaminated water or food. There are four different HEV genotypes in humans, with genotypes 1 and 2 being the most widespread. Genotypes 3 and 4 are found in animals and can also infect humans. Genotype 4 is prevalent in Asia, mainly in China. In Italy, only one outbreak of HEV-4 has been documented, which occurred in 2011, involving five patients. In 2013, HEV G4 was also detected in a pig farm. Since then, no further evidence of HEV genotype 4 has been found in the country. This study describes the first detection of HEV genotype 4, subtype d, in wastewater in central Italy, despite a lack of any clinical case reported in the area. By using a multiplex PCR protocol and two sequencing strategies, Illumina and ONT, the virus's complete genome was sequenced and characterized as subtype 4d. These findings shed light on the potential of environmental surveillance for infectious agents to improve our understanding of epidemiology and support public health efforts.

A chance-constrained stochastic chiller sequencing strategy considering life-expectancy of chiller plant

This article focuses on addressing the chiller sequencing problem of chiller plants by establishing a comprehensive energy management framework. The contribution of this work is threefold. Firstly, a distributive modeling architecture is presented that establishes five concurrent models as input to the framework. A synergy among these models is exploited to formulate a chance-constrained stochastic chiller sequencing problem. Secondly, a quantified life expectancy model of the chiller plant is introduced and a case is made for why it is influential in delivering industrially applicable solutions. The model attempts to strike a balance between economic optimality and improved reliability. Thirdly, a chiller data pre-processing protocol incubating two heuristic algorithms is proposed to address measurement uncertainties of the chiller plant state variables. Furthermore, this work develops a robust ensemble model to accurately forecast the cooling load and embeds a chain of reformulations to improve the global solution's optimality. The developed framework is realized with the plant at the Indian Institute of Technology Gandhinagar. The results confirm that the proposed framework leads to a significant amount of power savings. In comparison to conventional scheduling, the chiller plant power consumption can be reduced by up to 6.2 %, thereby illustrating its efficacy.

Targeted sequencing identifies risk variants in 202 candidate genes for neurodevelopmental disorders

With the continuous deepening of genetic research on neurodevelopmental disorders (NDDs), more patients have been identified the causal or candidate genes. However, it is still urgent needed to increase the sample size to confirm the associations between variants and clinical manifestations. We previously performed molecular inversion probe sequencing of autism spectrum disorder (ASD) candidate genes in 1543 ASD patients. In this study, we used the same method to detect de novo variants (DNVs) in 665 NDD patients with intellectual disability (ID) and/or epilepsy (EP) for genetic analysis and diagnosis. We compared findings from ID/EP and ASD patients to improve our understanding of different subgroups of NDDs. We identified 72 novel variants and 39 DNVs. A totally of 5.71 % (38/665) of the patients were genetically diagnosed by this sequencing strategy. ID/EP patients demonstrated a higher prevalence of likely gene disruptive DNVs in ASD genes than the healthy population. Regarding high-risk genes, SCN1A and CKDL5 were more frequently mutated in ID/EP patients than in ASD patients. Our data provide an overview of the mutation burden in ID/EP patients from the perspective of high risk ASD genes, indicating the differences and association of NDDs subgroups.