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Environmental studies often require culture and characterization to understand the prevalence, distribution, persistence and functions of target microorganisms in ecological habitats. Isolating pure microbiological monocultures allows the phenotypic characterization of microorganisms to study their functional properties. For efficient isolation of low-prevalence organisms, enrichment followed by PCR screening is performed to identify positive samples for subsequent culture. Molecular characterization, strain-typing, and genotyping of isolated microorganisms is best comprehensively performed using whole-genome sequencing. This article outlines end-to-end protocols for screening, isolation, and sequencing of microbes from environmental samples. We provide systematic methods for environmental study design, enrichment, screening, and isolation of target microorganisms. Species identification is performed using qPCR or MALDI-TOF MS. Genomic DNA is extracted for whole-genome sequencing using the Oxford Nanopore platform. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Designing and conducting an environmental soil sampling study Basic Protocol 2: Enrichment of microbes from environmental soil samples Alternate Protocol 1: Collection and enrichment of microbes from environmental water samples Basic Protocol 3: Screening of enriched samples by direct qPCR Basic Protocol 4: Enumeration and isolation of enriched samples using selective medium Basic Protocol 5: Species confirmation using colony qPCR Alternate Protocol 2: Species identification using a MALDI-TOF MS Biotyper Alternate Protocol 3: Species identification of bacterial isolates using universal PCR primers and Sanger sequencing Basic Protocol 6: Cryostorage of bacterial isolates Basic Protocol 7: Extraction of genomic DNA Basic Protocol 8: Quality check of extracted genomic DNA Basic Protocol 9: Whole-genome sequencing using the Oxford Nanopore MinION Platform.

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Atopic dermatitis and abnormalities in tooth development (including hypomineralization, hypodontia and microdontia) have been observed to co-occur in some patients. A common pathogenesis pathway that involves genes and protein interactions has been hypothesized. This review aims to first provide a description of the key gene mutations and signaling pathways associated with atopic dermatitis and tooth agenesis (i.e., the absence of teeth due to developmental failure) and identify the possible association between the two diseases. Second, utilizing a list of genes most commonly associated with the two diseases, we conducted a protein-protein network interaction analysis using the STRING database and identified a novel association between the Wnt/ß-catenin signaling pathway (major pathway responsible for TA) and desmosomal proteins (component of skin barrier that affect the pathogenesis of AD). Further investigation into the mechanisms that may drive their co-occurrence and underlie the development of the two diseases is warranted.

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Rapid and sensitive Escherichia coli (E. coli) detection is important in determining environmental contamination, food contamination, as well as bacterial infection. Conventional methods based on bacterial culture suffer from long testing time (24 h), whereas novel nucleic acid-based and immunolabelling approaches are hindered by complicated operation, the need of complex and costly equipment, and the lack of differentiation of live and dead bacteria. Herein, we propose a chemiluminescence digital microwell array chip based on the hydrolysis of 6-Chloro-4-methylumbelliferyl-ß-D-glucuronide by the ß-D-glucuronidase in E. coli to achieve fast single bacterial fluorescence detection. Taking the advantage of the picoliter microwells, single bacteria are digitally encapsulated in these microwells, thus the accurate quantification of E. coli can be realized by counting the number of positive microwells. We also show that the chemiluminescence digital microwell array chip is not affected by the turbidity of the test samples as well as the temperature. Most importantly, our method can differentiate live and dead bacteria through bacterial proliferation and enzyme expression, which is confirmed by detecting E. coli after pH and chlorination treatment. By comparing with the standard method of plate counting, our method has comparable performance but significantly reduces the testing time from over 24 h-2 h and 4 h for qualitative and quantitative analysis, respectively. In addition, the microfluidic chip is portable and easy to operate without external pump, which is promising as a rapid and on-site platform for single E. coli analysis in water and food monitoring, as well as infection diagnosis.

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Here, we report the genome of ESBL-producing Klebsiella pneumoniae strain C43, which was isolated from an environmental water sample. The genome is 5,614,412 bp in size with GC content of 56.86% with multidrug antimicrobial resistance genes and several metal resistance gene operons.

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With the advent of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, several vaccines have been developed to mitigate its spread and prevent adverse consequences of the Coronavirus Disease 2019 (COVID-19). The mRNA technology is an unprecedented vaccine, usually given in two doses to prevent SARS-CoV-2 infections. Despite effectiveness and safety, inter-individual immune response heterogeneity has been observed in recipients of mRNA-based vaccines. As a novel disease, the specific immune response mechanism responsible for warding off COVID-19 remains unclear at this point. However, significant evidence suggests that humoral response plays a crucial role in affording immunoprotection and preventing debilitating sequelae from COVID-19. As such, this paper focused on the possible effects of age, sex, serostatus, and comorbidities on humoral response (i.e. total antibodies, IgG, and/or IgA) of different populations post-mRNA-based Pfizer-BioNTech vaccination. A systematic search of literature was performed through PubMed, Cochrane CENTRAL, Google Scholar, Science Direct, medRxiv, and Research Square. Studies were included if they reported humoral response to COVID-19 mRNA vaccines. A total of 32 studies were identified and reviewed, and the percent differences of means of reported antibody levels were calculated for comparison. Findings revealed that older individuals, male sex, seronegativity, and those with more comorbidities mounted less humoral immune response. Given these findings, several recommendations were proposed regarding the current vaccination practices. These include giving additional doses of vaccination for immunocompromised and elderly populations. Another recommendation is conducting clinical trials in giving a combined scheme of mRNA vaccines, protein vaccines, and vector-based vaccines.

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Whole-genome sequencing of prokaryotes is now readily available and affordable on next-generation sequencing platforms. However, the process of de novo assembly can be complicated and tedious for those without a background in computational biology, bioinformatics, or UNIX. Licenses for commercial bioinformatics software may be costly and limited in flexibility. GALAXY is a powerful graphical open-source code-free bioinformatics platform that is freely available on multiple public and private servers. Here, we describe a bacterial de novo assembly workflow using GALAXY. It performs de novo genome assembly using short reads, long reads, or a hybrid method using both short and long reads. Genome annotation, prediction of antimicrobial resistance genes, and multi-locus sequence typing are subsequently performed to characterize the draft genome. Performing genome assembly and annotation on this pipeline allows documentation, parameterization, and sharing, facilitating replication, reuse, and reproducibility of both data and methods. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Quality check of NGS reads Basic Protocol 2: De novo assembly using Unicycler Basic Protocol 3: Assembly quality check using QUAST and Bandage Basic Protocol 4: Genome annotation using Prokka Basic Protocol 5: Prediction of antimicrobial resistance genes (ARGs) Basic Protocol 6: Multi-locus sequence typing (MLST).

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Here, we report the genome sequence of Enterobacter hormaechei subsp. steigerwaltii strain BEI01, originally deposited as a member of the Enterobacter cloacae complex. The genome is 4,900,246 bp in size with a GC content of 55.44%; it contains multidrug antimicrobial resistance genes and several metal resistance gene operons.

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There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting.

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Fibroblast growth factor 21 (FGF21) and adiponectin increase the expression of genes involved in antioxidant pathways, but their roles in mediating oxidative stress and arterial stiffness with ageing and habitual exercise remain unknown. We explored the role of the FGF21-adiponectin axis in mediating oxidative stress and arterial stiffness with ageing and habitual exercise. Eighty age- and sex-matched healthy individuals were assigned to younger sedentary or active (18-36 years old, n = 20 each) and older sedentary or active (45-80 years old, n = 20 each) groups. Arterial stiffness was measured indirectly using pulse wave velocity (PWV). Fasted plasma concentrations of FGF21, adiponectin and oxidized low-density lipoprotein (oxLDL) were measured. PWV was 0.2-fold higher and oxLDL concentration was 25.6% higher (both p < 0.001) in older than younger adults, despite no difference in FGF21 concentration (p = 0.097) between age groups. PWV (p = 0.09) and oxLDL concentration (p = 0.275) did not differ between activity groups but FGF21 concentration was 9% lower in active than sedentary individuals (p = 0.011). Adiponectin concentration did not differ by age (p = 0.642) or exercise habits (p = 0.821). In conclusion, age, but not habitual exercise, was associated with higher oxidative stress and arterial stiffness. FGF21 and adiponectin did not differ between younger and older adults, meaning that it is unlikely that they mediate oxidative stress and arterial stiffness in healthy adults.

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What is the central question of this study? Fibroblast growth factor 21 (FGF21) plays important therapeutic roles in metabolic diseases but is associated with bone loss, through insulin-like growth factor binding protein 1 (IGFBP1), in animals. However, the effect of the FGF21-IGFBP1 axis on age-related bone loss has not been explored in humans. What is the main finding and its importance? Using 'genetically linked' parent and child family pairs, we show that the FGF21 concentration, but not the IGFBP1 concentration, is higher in older than in younger adults. Our results suggest that age-associated decline in bone mineral density is associated with FGF21 and increased bone turnover but not likely to involve IGFBP1 in healthy humans. ABSTRACT: Bone fragility increases with age. The fibroblast growth factor 21 (FGF21)-insulin-like growth factor binding protein 1 (IGFBP1) axis regulates bone loss in animals. However, the role of FGF21 in mediating age-associated bone fragility in humans remains unknown. The purpose of this study was to explore the FGF21-regulatory axis in bone turnover and the age-related decline in bone mineral density (BMD). Twenty 'genetically linked' family (parent and child) pairs were recruited. Younger adults were 22-39 years old and older adults 60-71 years old. The BMD and serum concentrations of FGF21, IGFBP1, receptor activator of nuclear factor-κB ligand (RANKL), tartrate-resistant acid phosphatase 5b (TRAP5b) and bone-specific alkaline phosphatase (BAP) were measured. Older adults had 10-18% lower BMD at the hip and spine (P < 0.008) and a twofold higher FGF21 concentration (P < 0.001). The IGFBP1 concentration was similar in younger and older adults (P = 0.961). The RANKL concentration was 44% lower (P = 0.006), whereas TRAP5b and BAP concentrations were 36 and 31% higher (P = 0.01 and P = 0.004), respectively, in older adults than in younger adults. Adjusting for sex did not affect these results. The FGF21 concentration was negatively correlated with BMD at the spine (r = -0.460, P = 0.003), but not with the IGFBP1 concentration (r = -0.144, P = 0.374). The IGFBP1 concentration was not correlated with BMD at the hip or spine (all P > 0.05). In humans, FGF21 might be involved in the age-associated decline in BMD, especially at the spine, through increased bone turnover. IGFBP1 is unlikely to be the downstream effector of FGF21 in driving the age-associated decline in BMD and in RANKL-associated osteoclast differentiation.

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INTRODUCTION: Aging increases the prevalence of glucose intolerance, but exercise improves glucose homeostasis. The fibroblast growth factor 21 (FGF21)-adiponectin axis helps regulate glucose metabolism. However, the role of FGF21 in mediating glucose metabolism with aging and exercise remains unknown. PURPOSE: This study examined whether FGF21 responses to a glucose challenge are associated with habitual exercise, aging and glucose regulation. METHODS: Eighty age- and sex-matched healthy individuals were assigned to young sedentary and active (≤36 yr, n = 20 each group) and older sedentary and active (≥45 yr, n = 20 each group) groups. Fasted and postprandial blood glucose concentration and plasma concentration of insulin, FGF21, and adiponectin were determined during an oral glucose tolerance test (OGTT). RESULTS: During the OGTT, glucose concentrations were 9% higher (P = 0.008) and FGF21 concentrations were 58% higher (P = 0.014) in the older than the younger group, independent of activity status. Active participants had 40% lower insulin concentration and 53% lower FGF21 concentration than sedentary participants, independent of age (all P < 0.001). Adiponectin concentration during the OGTT did not differ by age (P = 0.448) or activity status (P = 0.611). Within the younger group, postprandial glucose, insulin and FGF21 concentrations during the OGTT were lower in active than in sedentary participants. In the older group, only postprandial insulin and FGF21 concentrations were lower in active participants. CONCLUSIONS: FGF21, but not adiponectin, response during the OGTT is higher in older than younger adults and lower in active than sedentary individuals. Exercise-associated reduction in OGTT glucose concentrations was observed in younger but not older adults.

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OBJECTIVE: To investigate age-associated changes in airway microbiome composition and their relationships with lung function and arterial stiffness among genetically matched young and elderly pairs. METHODS: Twenty-four genetically linked family pairs comprised of younger (≤40 years) and older (≥60 years) healthy participants were recruited (Total n = 48). Lung function and arterial stiffness (carotid-femoral pulse wave velocity (PWV) and augmentation index (AIx)) were assessed. Sputum samples were collected for targeted 16S rRNA gene amplicon sequencing and correlations between microbiome composition, lung function and arterial stiffness were investigated. RESULTS: Elderly participants exhibited reductions in lung function (FEV1 (p<0.001), FVC (p<0.001) and percentage FEV1/FVC (p = 0.003)) and a 1.3-3.9-fold increase in arterial stiffness (p<0.001) relative to genetically related younger adults. Elderly adults had a higher relative abundance of Firmicutes (p = 0.035) and lower relative abundance of Proteobacteria (p = 0.014), including specific genera Haemophilus (p = 0.024) and Lautropia (p = 0.020) which were enriched in the younger adults. Alpha diversity was comparable between young and elderly pairs (p>0.05) but was inversely associated with lung function (FEV1%Predicted and FVC %Predicted) in the young (p = 0.006 and p = 0.003) though not the elderly (p = 0.481 and p = 0.696). Conversely, alpha diversity was negatively associated with PWV in the elderly (p = 0.01) but not the young (p = 0.569). Specifically, phylum Firmicutes including the genus Gemella were correlated with lung function (FVC %Predicted) in the young group (p = 0.047 and p = 0.040), while Fusobacteria and Leptotrichia were associated with arterial stiffness (PWV) in the elderly (both p = 0.004). CONCLUSION: Ageing is associated with increased Firmicutes and decreased Proteobacteria representation in the airway microbiome among a healthy Asian cohort. The diversity and composition of the airway microbiome is independently associated with lung function and arterial stiffness in the young and elderly groups respectively. This suggests differential microbial associations with these phenotypes at specific stages of life with potential prognostic implications.

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The Asia Pacific region, home to two-thirds of the world's population and ten of the least developed countries, is considered a regional hot-spot for the emergence and spread of antimicrobial resistance (AMR). Despite this, there is a dearth of high-quality regional data on the extent of AMR. Recognising the urgency to close this gap, Singapore organised a meeting to discuss the problems in the region and frame a call for action. Representatives from across the region and beyond attended the meeting on the "Antimicrobial Resistance in the Asia Pacific & its impact on Singapore" held in November 2018. This meeting report is a summary of the discussions on the challenges and progress in surveillance, drivers and levers of AMR emergence, and the promising innovations and technologies that could be used to combat the increasing threat of AMR in the region. Enhanced surveillance and research to provide improved evidence-based strategies and policies are needed. The major themes that emerged for an action plan are working towards a tailored solution for the region by harnessing the One Health approach, enhancing inter-country collaborations, and collaboratively leverage upon new emerging technologies. A regionally coordinated effort that is target-driven, sustainable and builds on a framework facilitating communication and governance will strengthen the fight against AMR in the Asia Pacific region.

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INTRODUCTION: Mutations within SCN5A are found in a significant proportion (15-30%) of Brugada syndrome (BrS) cases and impair sodium transport across excitable cardiac cells that mediate ventricular contractions. Genetic testing offers a means to clinically assess and manage affected individuals and their family members. METHODS AND RESULTS: The proband at age 44 years old exhibited a syncopal event during exercise, and presented later with a spontaneous type-I BrS pattern on 12­lead resting electrocardiogram (ECG). Mutational analysis performed across all SCN5A exons revealed a unique three base-pair deletion p.M741_T742delinsI (c.2223_2225delGAC), in a heterozygous state in the proband and 2 siblings. This mutation was not seen in a cohort of 105 ethnicity-matched controls or in public genome databases. Patch clamp electrophysiology study conducted in TSA201 cells showed an abolishment of sodium current (INa). The proband, and several relatives, also harboured a known SCN5A variant, p.R1193Q (c.3578G>A). CONCLUSION: Our study has demonstrated the deleterious effect of a novel SCN5A mutation p.M741_T742delinsI (c.2223_2225delGAC). The findings highlight the complex effects of gender and age in phenotype manifestation. It also offers insights into improving the long-term management of BrS, and the utility of cascade genetic screening for risk stratification.

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Environmental Burkholderia pseudomallei has been postulated to be aerosolized during ploughing and heavy rain, and could result in inhalational melioidosis. Here, we determined the presence of B. pseudomallei in soil, paddy field water (PFW), air, and rainwater samples in a single rice paddy field in Ubon Ratchathani, northeast Thailand. In 2012, we collected 100 soil samples during the dry season, 10 PFW samples during the monsoon season, 77 air samples during ploughing (N = 31) and heavy rains (N = 46), and 60 rainwater samples during 12 rain events. We found that 32 soil samples (32%), six PFW samples (60%), and none of the air and rainwater samples were culture positive for B. pseudomallei. Other soil bacteria were isolated from air and rainwater samples. Mean quantitative count of B. pseudomallei estimated from two culture-positive PFW samples was 200 colony forming units/mL. Our findings suggest that the risk of melioidosis acquisition by inhalation in Thailand might be low.

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The human amylase gene locus at chromosome 1p21.1 is structurally complex. This region contains two pancreatic amylase genes, AMY2B, AMY2A, and a salivary gene AMY1. The AMY1 gene harbors extensive copy number variation (CNV), and recent studies have implicated this variation in adaptation to starch-rich diets and in association to obesity for European and Asian populations. In this study, we showed that by combining quantitative PCR and digital PCR, coupled with careful experimental design and calibration, we can improve the resolution of genotyping CNV with high copy numbers (CNs). In two East Asian populations of Chinese and Malay ethnicity studied, we observed a unique non-normal distribution of AMY1 diploid CN genotypes with even:odd CNs ratio of 4.5 (3.3-4.7), and an association between the common AMY2A CN = 2 genotype and odd CNs of AMY1, that could be explained by the underlying haplotypic structure. In two further case-control cohorts (n = 932 and 145, for Chinese and Malays, respectively), we did not observe the previously reported association between AMY1 and obesity or body mass index. Improved methods for accurately genotyping multiallelic CNV loci and understanding the haplotype complexity at the AMY1 locus are necessary for population genetics and association studies.

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Using three-dimensional printing, we produced adaptors to attach a smartphone with camera to the eyepiece of the Airtraq(®) laryngoscope. This low-cost system enabled a team to simultaneously view the laryngoscopy process on the smartphone screen, and also enabled image transmission. We compared the Airtraq(®) with the smartphone Airtraq(®) system in a crossover study of trainee anesthesiologists performing tracheal intubation in a manikin. We also evaluated the smartphone Airtraq(®) system for laryngoscopy and tracheal intubation in 30 patients, including image transmission to and communication with a remote instructor. In the manikin study, the smartphone Airtraq(®) system enabled instruction where both trainee and instructor could view the larynx simultaneously, and did not substantially increase the time required for intubation. In the patient study, we were able to view the larynx in all 30 patients, and the remote instructor was able to receive the images and to respond on correctness of laryngoscopy and tracheal tube placement. Tracheal intubation was successful within 90s in 19 (63 %) patients. In conclusion, use of a smartphone with the Airtraq(®) may facilitate instruction and communication of laryngoscopy with the Airtraq(®), overcoming some of its limitations.

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AIMS: Brugada syndrome (BrS) is a rare heritable ventricular arrhythmia. Genetic defects in SCN5A, a gene that encodes the α-subunit of the sodium ion channel Nav1.5, are present in 15-30% of BrS cases. SCN5A remains by far, the highest yielding gene for BrS. We studied a young male who presented with syncope at age 11. This proband was screened for possible disease causing SCN5A mutations. The inheritance pattern was also examined amongst his first-degree family members. METHODS AND RESULTS: The proband had a baseline electrocardiogram that showed Type 2 BrS changes, which escalated to a characteristic Type I BrS pattern during a treadmill test before polymorphic ventricular tachycardia onset at a cycle length of 250 ms. Mutational analysis across all 29 exons in SCN5A of the proband and first-degree relatives of the family revealed that the proband inherited a compound heterozygote mutation in SCN5A, specifically p.A226V and p.R1629X from each parent. To further elucidate the functional changes arising through these mutations, patch-clamp electrophysiology was performed in TSA201 cells expressing the mutated SCN5A channels. The p.A226V mutation significantly reduced peak sodium current (INa) to 24% of wild type (WT) whereas the p.R1629X mutation abolished the current. To mimic the functional state in our proband, functional expression of the compound variants A226V + R1629X resulted in overall peak INa of only 13% of WT (P < 0.01). CONCLUSION: Our study is the first to report a SCN5A compound heterozygote in a Singaporean Chinese family. Only the proband carrying both mutations displayed the BrS phenotype, thus providing insights into the expression and penetrance of BrS in an Asian setting.

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Severe myopia (defined as spherical equivalent < -6.0 D) is a predominant problem in Asian countries, resulting in substantial morbidity. We performed a meta-analysis of four genome-wide association studies (GWAS), all of East Asian descent totaling 1603 cases and 3427 controls. Two single nucleotide polymorphisms (SNPs) (rs13382811 from ZFHX1B [encoding for ZEB2] and rs6469937 from SNTB1) showed highly suggestive evidence of association with disease (P < 1 × 10(-7)) and were brought forward for replication analysis in a further 1241 severe myopia cases and 3559 controls from a further three independent sample collections. Significant evidence of replication was observed, and both SNP markers surpassed the formal threshold for genome-wide significance upon meta-analysis of both discovery and replication stages (P = 5.79 × 10(-10), per-allele odds ratio (OR) = 1.26 for rs13382811 and P = 2.01 × 10(-9), per-allele OR = 0.79 for rs6469937). The observation at SNTB1 is confirmatory of a very recent GWAS on severe myopia. Both genes were expressed in the human retina, sclera, as well as the retinal pigmented epithelium. In an experimental mouse model for myopia, we observed significant alterations to gene and protein expression in the retina and sclera of the unilateral induced myopic eyes for Zfhx1b and Sntb1. These new data advance our understanding of the molecular pathogenesis of severe myopia.

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Whole-genome sequencing across multiple samples in a population provides an unprecedented opportunity for comprehensively characterizing the polymorphic variants in the population. Although the 1000 Genomes Project (1KGP) has offered brief insights into the value of population-level sequencing, the low coverage has compromised the ability to confidently detect rare and low-frequency variants. In addition, the composition of populations in the 1KGP is not complete, despite the fact that the study design has been extended to more than 2,500 samples from more than 20 population groups. The Malays are one of the Austronesian groups predominantly present in Southeast Asia and Oceania, and the Singapore Sequencing Malay Project (SSMP) aims to perform deep whole-genome sequencing of 100 healthy Malays. By sequencing at a minimum of 30× coverage, we have illustrated the higher sensitivity at detecting low-frequency and rare variants and the ability to investigate the presence of hotspots of functional mutations. Compared to the low-pass sequencing in the 1KGP, the deeper coverage allows more functional variants to be identified for each person. A comparison of the fidelity of genotype imputation of Malays indicated that a population-specific reference panel, such as the SSMP, outperforms a cosmopolitan panel with larger number of individuals for common SNPs. For lower-frequency (<5%) markers, a larger number of individuals might have to be whole-genome sequenced so that the accuracy currently afforded by the 1KGP can be achieved. The SSMP data are expected to be the benchmark for evaluating the value of deep population-level sequencing versus low-pass sequencing, especially in populations that are poorly represented in population-genetics studies.

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