To Analyze the Sensitivity of RT-PCR Assays Employing S Gene Target Failure with Whole Genome Sequencing Data during Third Wave by SARS-CoV-2 Omicron Variant

Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.

Safety evaluation and whole genome sequencing for revealing the ability of Penicillium oxalicum WX-209 to safely and effectively degrade citrus segments

The microbial potential of Penicillium has received critical attention.The present research aimed to elucidate the efficacy of crude enzyme secreted from Penicillium oxalicum WX-209 in degrading citrus segments and evaluate the safety of the process.Results showed that citrus segment membranes gradually dissolved after treatment with the crude enzyme solution,indicating good degradation capability.No significant differences in body weight,food ingestion rate,hematology,blood biochemistry,and weight changes of different organs were found between the enzyme intake and control groups.Serial experiments showed that the crude enzyme had high biological safety.Moreover,the whole genome of P.oxalicum WX-209 was sequenced by PacBio and Illumina platforms.Twenty-five scaffolds were assembled to generate 36 Mbp size of genome sequence comprising 11369 predicted genes modeled with a GC content of 48.33%.A total of 592 genes were annotated to encode enzymes related to carbohydrates,and some degradation enzyme genes were identified in strain P.oxalicum WX-209.

Surveillance of emerging SARS-CoV-2 variants by nanopore technology-based genome sequencing

Objective:To surveill emerging variants by nanopore technology-based genome sequencing in different COVID-19 waves in Sri Lanka and to examine the association with the sample characteristics,and vaccination status.Methods:The study analyzed 207 RNA positive swab samples received to sequence laboratory during different waves.The N gene cut-off threshold of less than 30 was considered as the major inclusion criteria.Viral RNA was extracted,and elutes were subjected to nanopore sequencing.All the sequencing data were uploaded in the publicly accessible database,GISAID.Results:The Omicron,Delta and Alpha variants accounted for 58%,22%and 4%of the variants throughout the period.Less than 1%were Kappa variant and 16%of the study samples remained unassigned.Omicron variant was circulated among all age groups and in all the provinces.Ct value and variants assigned percentage was 100%in Ct values of 10-15 while only 45%assigned Ct value over 25.Conclusions:The present study examined the emergence,prevalence,and distribution of SARS-CoV-2 variants locally and has shown that nanopore technology-based genome sequencing enables whole genome sequencing in a low resource setting country.

Genome sequencing provides potential strategies for drug discovery and synthesis

Medicinal plants are renowned for their abundant production of secondary metabolites,which exhibit notable pharmacological activities and great potential for drug development.The biosynthesis of secondary metabolites is highly intricate and influenced by various intrinsic and extrinsic factors,resulting in substantial species diversity and content variation.Consequently,precise regulation of secondary metabolite synthesis is of utmost importance.In recent years,genome sequencing has emerged as a valuable tool for investigating the synthesis and regulation of secondary metabolites in medicinal plants,facilitated by the widespread use of high-throughput sequencing technologies.This review highlights the latest advancements in genome sequencing within this field and presents several strategies for studying secondary metabolites.Specifically,the article elucidates how genome sequencing can unravel the pathways for secondary metabolite synthesis in medicinal plants,offering insights into the functions and regulatory mechanisms of participating enzymes.Comparative analyses of plant genomes allow identification of shared pathways of metabolite synthesis among species,thereby providing novel avenues for obtaining cost-effective biosynthetic intermediates.By examining individual genomic variations,genes or gene clusters associated with the synthesis of specific compounds can be discovered,indicating potential targets and directions for drug development and the exploration of alternative compound sources.Moreover,the advent of gene-editing technology has enabled the precise modifications of medicinal plant genomes.Optimization of specific secondary metabolite synthesis pathways becomes thus feasible,enabling the precise editing of target genes to regulate secondary metabolite production within cells.These findings serve as valuable references and lessons for future drug development endeavors,conservation of rare resources,and the exploration of new resources.

The evolution of cancer genomic medicine in Japan and the role of the National Cancer Center Japan

The journey to implement cancer genomic medicine(CGM)in oncology practice began in the 1980s,which is considered the dawn of genetic and genomic cancer research.At the time,a variety of activating oncogenic alterations and their functional significance were unveiled in cancer cells,which led to the development of molecular targeted therapies in the 2000s and beyond.Although CGM is still a relatively new discipline and it is difficult to predict to what extent CGM will benefit the diverse pool of cancer patients,the National Cancer Center(NCC)of Japan has already contributed considerably to CGM advancement for the conquest of cancer.Looking back at these past achievements of the NCC,we predict that the future of CGM will involve the following:1)A biobank of paired cancerous and non-cancerous tissues and cells from various cancer types and stages will be developed.The quantity and quality of these samples will be compatible with omics analyses.All biobank samples will be linked to longitudinal clinical information.2)New technologies,such as whole-genome sequencing and artificial intelligence,will be introduced and new bioresources for functional and pharmacologic analyses(e.g.,a patient-derived xenograft library)will be systematically deployed.3)Fast and bidirectional translational research(bench-to-bedside and bedside-to-bench)performed by basic researchers and clinical investigators,preferably working alongside each other at the same institution,will be implemented;4)Close collaborations between academia,industry,regulatory bodies,and funding agencies will be established.5)There will be an investment in the other branch of CGM,personalized preventive medicine,based on the individual's genetic predisposition to cancer.

Identification of novel genes associated with atherosclerosis in Bama miniature pig

Background:Atherosclerosis is a chronic cardiovascular disease of great concern.However,it is difficult to establish a direct connection between conventional small animal models and clinical practice.The pig's genome,physiology,and anatomy reflect human biology better than other laboratory animals,which is crucial for studying the pathogenesis of atherosclerosis.Methods:We used whole-genome sequencing data from nine Bama minipigs to perform a genome-wide linkage analysis,and further used bioinformatic tools to filter and identify underlying candidate genes.Candidate gene function prediction was performed using the online prediction tool STRING 12.0.Immunohistochemistry and immunofluorescence were used to detect the expression of proteins encoded by candidate genes.Results:We mapped differential single nucleotide polymorphisms(SNPs)to genes and obtained a total of 102 differential genes,then we used GO and KEGG pathway enrichment analysis to identify four candidate genes,including SLA-1,SLA-2,SLA-3,and TAP2.nsSNPs cause changes in the primary and tertiary structures of SLA-I and TAP2 proteins,the primary structures of these two proteins have undergone amino acid changes,and the tertiary structures also show slight changes.In addition,immunohistochemistry and immunofluorescence results showed that the expression changes of TAP2 protein in coronary arteries showed a trend of increasing from the middle layer to the inner layer.Conclusions:We have identified SLA-I and TAP2 as potential susceptibility genes of atherosclerosis,highlighting the importance of antigen processing and immune response in atherogenesis.

Isolation and Characterization of SARS-CoV-2 in Kenya

The discovery of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) in Wuhan, Hubei province, China, in December 2019 raised global health warnings. Quickly, in 2020, the virus crossed borders and infected individuals across the world, evolving into the COVID-19 pandemic. Notably, early signs of the virus’s existence were observed in various countries before the initial outbreak in Wuhan. As of 12th of April, the respiratory disease had infected over 762 million people worldwide, with over 6.8 million deaths recorded. This has led scientists to focus their efforts on understanding the virus to develop effective means to diagnose, treat, prevent, and control this pandemic. One of the areas of focus is the isolation of this virus, which plays a crucial role in understanding the viral dynamics in the laboratory. In this study, we report the isolation and detection of locally circulating SARS-CoV-2 in Kenya. The isolates were cultured on Vero Cercopithecus cell line (CCL-81) cells, RNA extraction was conducted from the supernatants, and reverse transcriptase-polymerase chain reaction (RT-PCR). Genome sequencing was done to profile the strains phylogenetically and identify novel and previously reported mutations. Vero CCL-81 cells were able to support the growth of SARS-CoV-2 in vitro, and mutations were detected from the two isolates sequenced (001 and 002). Genome sequencing revealed the circulation of two isolates that share a close relationship with the Benin isolate with the D614G common mutation identified along the S protein. These virus isolates will be expanded and made available to the Kenya Ministry of Health and other research institutions to advance SARS-CoV-2 research in Kenya and the region.

The Application of Nicotiana benthamiana as a Transient Expression Host to Clone the Coding Sequences of Plant Genes

Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patterns in vivo. These challenges require the development of alternative CDS cloning technologies. In this study, we found that the genomic intron-containing gene coding sequences (gDNA) from Arabidopsis thaliana, Oryza sativa, Brassica napus, and Glycine max can be correctly transcribed and spliced into mRNA in Nicotiana benthamiana. In contrast, gDNAs from Triticum aestivum and Sorghum bicolor did not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from the N. benthamiana leaves, making it conducive to the cloning of CDS target genes. Our data demonstrate that N. benthamiana can be used as an effective host for the cloning CDS of plant genes.

Epidemiology and Clonal Spread Evidence of Carbapenem-Resistant Organisms in the Center of Care and Protection of Orphaned Children, Vietnam

Objective: To determine the prevalence of colonization and transmission of carbapenem-resistant Gram-negative organisms in order to develop of an effective infection prevention program. Design: Cross-sectional study with carbapenem-resistant organisms (CRO) colonization detection from the fecal specimens of 20 Health Care Workers (HCWs) and 67 residents and 175 random environment specimens from September 2022 to September 2023. Setting: A Care and Protection Centre of Orphaned Children in South of HCM City. Participants: It included 20 HCWs, 67 residents, and 175 randomly collected environmental specimens. Method: Rectal and environmental swabs were collected from 20 HCWs, 67 residents (most of them were children), and 175 environmental specimens. MELAB Chromogenic CARBA agar plates, Card NID, and NMIC-500 CPO of the BD Phoenix TM Automated Microbiology System and whole genome sequencing (WGS) were the tests to screen, confirm CROs, respectively and determine CRO colonization and transmission between HCWs, residents, and the environment. Result: We detected 36 CRO isolates, including 6, 11 and 19 CROs found in 6 HCWs, 10 residents and 19 environments. The prevalence of detectable CRO was 30% (6/20) in HCWs, 14.92% (10/67) in residents, and 10.86% (19/175) in environmental swabs in our study. WGS demonstrated CRO colonization and transmission with the clonal spread of E. coli and A. nosocomialis, among HCWs and residents (children). Conclusion: Significant CRO colonization and transmission was evident in HCWs, residents, and the center environment. Cleaning and disinfection of the environment and performing regular hand hygiene are priorities to reduce the risk of CRO colonization and transmission.

High-throughput Sequencing Technology and Its Application

Gene sequencing is a great way to interpret life, and high-throughput sequencing technology is a revolutionary technological innovation in gene sequencing researches. This technology is characterized by low cost and high-throughput data. Currently, high-throughput sequencing technology has been widely applied in multi-level researches on genomics, transcriptomics and epigenomics. And it has fundamentally changed the way we approach problems in basic and translational researches and created many new possibilities. This paper presented a general description of high-throughput sequencing technology and a comprehensive review of its application with plain, concisely and precisely. In order to help researchers finish their work faster and better, promote science amateurs and understand it easier and better.

The role of bone marrow-derived cells in the origin of liver cancer revealed by single-cell sequencing

Objective:Epithelial cancers often originate from progenitor cells,while the origin of hepatocellular carcinoma(HCC)is still controversial.HCC,one of the deadliest cancers,is closely linked with liver injuries and chronic inflammation,which trigger massive infiltration of bone marrow-derived cells(BMDCs)during liver repair.Methods:To address the possible roles of BMDCs in HCC origination,we established a diethylnitrosamine(DEN)-induced HCC model in bone marrow transplanted mice.Immunohistochemistry and frozen tissue immunofluorescence were used to verify DENinduced HCC in the pathology of the disease.The cellular origin of DEN-induced HCC was further studied by single cell sequencing,single-cell nested PCR,and immunofluorescence-fluorescence in situ hybridization.Results:Studies by using single cell sequencing and biochemical analysis revealed that HCC cells in these mice were coming from donor mice BMDCs,and not from recipient mice.Furthermore,the copy numbers of mouse orthologs of several HCC-related genes previously reported in human HCC were also altered in our mouse model.DEN-induced HCCs exhibited a similar histological phenotype and genomic profile as human HCCs.Conclusions:These results suggested that BMDCs are an important origin of HCC,which provide important clues to HCC prevention,detection,and treatments.

QTL mapping of drought tolerance traits in soybean with SLAF sequencing

Drought stress is an important factor affecting soybean yield.Improving drought tolerance of soybean varieties can increase yield and yield stability when the stress occurs.Identifying QTL related to drought tolerance using molecular marker-assisted selection is able to facilitate the development of drought-tolerant soybean varieties.In this study,we used a high-yielding and drought-sensitive cultivar‘Zhonghuang 35’and a drought-tolerant cultivar‘Jindou 21’to establish F6:9 recombinant inbred lines.We constructed a highdensity genetic map using specific locus amplified fragment sequencing(SLAF-Seq)technology.The genetic map contained 8078 SLAF markers distributing across 20 soybean chromosomes with a total genetic distance of 3780.98 c M and an average genetic distance of0.59 c M between adjacent markers.Two treatments(irrigation and drought)were used in the field tests,the Additive-Inclusive Composite Interval Mapping(ICIM-ADD)was used to call QTL,and plant height and seed weight per plant were used as the indicators of drought tolerance.We identified a total of 23 QTL related to drought tolerance.Among them,seven QTL(q PH2,q PH6,q PH7,q PH17,q PH19-1,q PH19-2,and q PH19-3)on chromosomes 2,6,7,17,and 19 were related to plant height,and five QTL(q SWPP2,q SWPP6,q SWPP13,q SWPP17,and q SWPP19)on chromosomes 2,6,13,17,and 19 were related to seed weight and could be considered as the major QTL.In addition,three common QTL(q PH6/q SWPP6,q PH17/q SWPP17,and q PH19-3/q SWPP19)for both plant height and seed weight per plant were located in the same genomic regions on the same chromosomes.Three(q PH2,q PH17,and q PH19-2)and four novel QTL(q SWPP2,q SWPP13,q SWPP17,and q SWPP19)were identified for plant height and seed weight per plant,respectively.Two pairs of QTL(q PH2/q SWPP2 and q PH17/q SWPP17)were also common for both plant height and seed weight per plant.These QTL and closely linked SLAF markers could be used to accelerate breeding for drought tolerant cultivars via MAS.

A Roadmap for Whitefly Genomics Research:Lessons from Previous Insect Genome Projects

Due to evolving molecular and informatics technologies,modern genome sequencing projects have more different characteristics than what most biologists have become accustomed to during the capillary-based sequencing era.In this paper,we explore the characteristics that made past insect genome projects successful and place them in the context of next-generation sequencing.By taking into account the intricacies of whitefly biology and the community,we present a roadmap for whitefly-omics,which focuses on the formation of an international consortium,deployment of informatic platforms and realistic generation of reference sequence data.

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer

Objective Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes. Methods In this proof-of-concept study using Enterovirus 71 （EV71） and Coxsackievirus A16 （CA16） strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing. Results Within the first minute （min） of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min （on average）, 99% identity was achieved for 10 CA16 strains in a single run. Conclusion MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.

Impact of next-generation sequencing on molecular diagnosis of inherited non-syndromic hearing loss

Hearing loss is one of the most common birth defects,with inherited genetic defects play an important role,contributing to about 60%of deafness occurring in infants.However,hearing impairment is genetically heterogeneous,with both common and rare forms occurring due to mutations in estimated 500 genes.Due to the large number and presumably low mutation frequencies of those genes,it would be highly expensive and time-consuming to address this issue by conventional gene-by-gene Sanger sequencing.Next-generation sequencing is a revolutionary technology that allows the simultaneous screening of mutations in a large number of genes.It is cost effective compared to classical strategies of linkage analysis and direct sequencing when the number or size of genes is large,and thus has become a highly efficient strategy for identifying novel causative genes and mutations involved in heritable disease.In this review, we describe major NGS methodologies currently used for genetic disorders and highlight applications of these technologies in studies of molecular diagnosis and the discovery of genes implicated in non-syndromic hearing loss.

Genome-wide mining, characterization, and development of microsatellite markers in Marsupenaeus japonicus by genome survey sequencing

The kuruma prawn, Marsupenaeus japonicus, is one of the most cultivated and consumed species of shrimp. However, very few molecular genetic/genomic resources are publically available for it. Thus, the characterization and distribution of simple sequence repeats(SSRs) remains ambiguous and the use of SSR markers in genomic studies and marker-assisted selection is limited. The goal of this study is to characterize and develop genome-wide SSR markers in M. japonicus by genome survey sequencing for application in comparative genomics and breeding. A total of 326 945 perfect SSRs were identified, among which dinucleotide repeats were the most frequent class(44.08%), followed by mononucleotides(29.67%), trinucleotides(18.96%), tetranucleotides(5.66%), hexanucleotides(1.07%), and pentanucleotides(0.56%). In total, 151 541 SSR loci primers were successfully designed. A subset of 30 SSR primer pairs were synthesized and tested in 42 individuals from a wild population, of which 27 loci(90.0%) were successfully amplified with specific products and 24(80.0%) were polymorphic. For the amplified polymorphic loci, the alleles ranged from 5 to 17(with an average of 9.63), and the average PIC value was 0.796. A total of 58 256 SSR-containing sequences had significant Gene Ontology annotation; these are good functional molecular marker candidates for association studies and comparative genomic analysis. The newly identified SSRs significantly contribute to the M. japonicus genomic resources and will facilitate a number of genetic and genomic studies, including high density linkage mapping, genome-wide association analysis, marker-aided selection, comparative genomics analysis, population genetics, and evolution.

Genomics of pancreatic ductal adenocarcinoma

Pancreatic cancer is one of the worst prognostic cancers because of the late diagnosis and the absence of effective treatment. Within all subtypes of this disease, ductal adenocarcinoma has the shortest survival time. In recent years,global genomics profiling allowed the identification of hundreds of genes that are perturbed in pancreatic cancer. The integration of different omics sources in the study of pancreatic cancer has revealed several molecular mechanisms, indicating the complex history of its development. However, validation of these genes as biomarkers for early diagnosis, prognosis or treatment efficacy is still incomplete but should lead to new approaches for the treatment of the disease in the future.

Sequencing and Phylogenetic Analysis of Potato virus Y Liaoning Isolate in China

Complete genome sequence of Potato virus Y Liaoning isolate （PVY-LN） causing tobacco vein necrosis symptoms were isolated from Liaoning Province in China. Genome sequences of PVY-LN was 9 714 nucleotides in length, excluding the 3′- terminal poly （A） tail. PVY-LN encodes a single long open reading frame （ORF） of polyprotein that is predicted to be cleaved into ten mature proteins by three viral proteases. No recombination can be predicted in PVY-LN sequences compared with that of the other PVY strains using Recombination Detection Programe v. 4.16 （RDP4）. Complete genome sequence comparison and phylogenetic analysis indicated that PVY-LN is closely related to PVY necrosis strain （PVYN）.

Evaluation of A Single-reaction Method for Whole Genome Sequencing of Influenza A Virus using Next Generation Sequencing

Objective To evaluate a single-reaction genome amplification method, the multisegment reverse transcription-PCR （M-RTPCR）, for its sensitivity to full genome sequencing of influenza A virus, and the ability to differentiate mix-subtype virus, using the next generation sequencing （NGS） platform. Methods Virus genome copy was quantified and seria（iy diluted to different titers, followed by amplification with the M-RTPCR method and sequencing on the NGS platform. Furthermore, we manually mixed two subtype viruses to different titer rate and amplified the mixed virus with the M-RTPCR protocol, followed by whole genome sequencing on the NGS platform. We also used clinical samples to test the method performance. Results The M-RTPCR method obtained complete genome of testing virus at 125 copies/reaction and determined the virus subtype at titer of 25 copies/reaction. Moreover, the two subtypes in the mixed virus could be discriminated, even though these two virus copies differed by 200-fold using this amplification protocol. The sensitivity of this protocol we detected using virus RNA was also confirmed with clinical samples containing Iow-titer virus. Conclusion The M-RTPCR is a robust and sensitive amplification method for whole genome sequencing of influenza A virus using NGS platform.

Sequencing of the Cultivated Tetraploid Cotton Genome-Gossypium hirsutum

Cotton is an important cash crop in the world,and it plays an irreplaceable role in China's national economy.Cultivated upland cotton(Gossypium hirsutum L.) represents 95% of the world's cotton production,but it has a complex allotetraploid genome that contains at least 30000 genes in 2500