Bước đầu ứng dụng phương pháp giải trình tự gen thế hệ mới trong phát hiện đa tác nhân ở trẻ có triệu chứng tiêu chảy cấp

Bệnh tiêu chảy có tỷ lệ nhiễm và tử vong cao trong nhóm trẻ dưới 5 tuổi, đặc biệt là ở những nước đang phát triển như Việt Nam. Vi rút rota là một tác nhân quan trọng gây tiêu chảy bên cạnh trên 20 tác nhân vi khuẩn, vi rút và ký sinh trùng khác đã được xác định. Tuy nhiên có tới 20% những ca bệnh không xác định được nguyên nhân. Nghiên cứu này nhằm sử dụng phương pháp giải trình tự gen thế hệ mới (Nextgeneration sequencing - NGS) để phát hiện những tác nhân mới trong những ca bệnh tiêu chảy ở trẻ em. Mẫu phân của trẻ uống vắc xin rota mắc tiêu chảy được thu thập và sàng lọc một số tác nhân phổ biến bằng realtime RT-PCR và ELISA. Đồng thời, 3-4 mẫu của trẻ em Việt Nam và Bỉ được trộn lại để phân tích đa tác nhân bằng NGS. 25 và 15 nhóm tác nhân được phát hiện tương ứng ở mẫu bệnh phẩm của trẻ em Việt Nam và Bỉ. Bên cạnh các tác nhân vi rút quen thuộc họ Picornavirdae, Caliciviridae, phương pháp đã phát hiện một số vi rút không thường xuất hiện như vi rút thuộc họ Parvoviridae, một số thực khuẩn thể (bacteriophage) của V.cholera, Samonella trong các ca tiêu chảy ở cả Việt Nam và Bỉ. Như vậy phương pháp giải trình tự gen thế hệ mới có thể dùng để phát hiện đa tác nhân, bao gồm cả các tác nhântiềm năng.

A clinical, laboratory, radiological, and microbiologicalstudy of pneumonia associated with covid-19 indicating person-to-person transmission

Background: Novel coronavirus outbreak that originated in Wuhan, province of China has now been declared as one of the deadliest pandemics inflicting humankind in last hundred years. Method: In the present study, we have inferred the clinical, laboratory, radiological, and microbiological findings of five patients in a family cluster who presented with unexplained pneumonia after coming back from overseas and touchdown right here in India on 1st March 2020 earlier than lockdown and another member of the family who didn’tvisit thiscountry. Results: From March 10, 2020, we enrolled a family of six patients who travelled to SingaporeonJanuary 10th 2020and returned on March 1st 2020. Of six family members who travelled to Singapore, five were recognised as affected with the radical coronavirus (COVID 19). Additionally, one family member, who did not travel to overseas also became infected with the virus post14 days of staying with four of the family members. Five family members (aged 30–55 years) presented with symptoms like fever, upper or lower respiratory tract symptoms, or diarrhoea, or a combination of these 3–6 days after exposure. Phylogenetic evaluation of these five subjects’ RT-PCR amplicons and two full genomes by nextgeneration sequencing presented that this is a novel coronavirus, which is closest to the severe acute respiratory syndrome (SARS)-related coronaviruses. Conclusion: Our findings are steady with person-to-person transmission of this novel coronavirus in hospital (nosocomial) and family settings, and the reports of infected travellers in other geographical regions.

Aislamiento y caracterización de una cepa temprana de SARS-CoV-2 durante la epidemia de 2020 en Medellín, Colombia

Introducción. El nuevo coronavirus causante de un brote de enfermedad respiratoria aguda en China en diciembre de 2019 se identificó como SARS-CoV-2. La enfermedad, denominada COVID-19, fue declarada pandemia por la Organización Mundial de la Salud (OMS). El primer caso de COVID-19 en Colombia se reportó el 6 de marzo de 2020; en este estudio se caracterizó un aislamiento temprano del virus SARS-CoV-2 de una muestra ecolectada en abril de 2020.Objetivos. Describir y caracterizar una cepa temprana a partir de un aislamiento de SARSCoV-2 durante la pandemia en Colombia.Materiales y métodos. Se obtuvo una muestra de un paciente con COVID-19 confirmada por qRT-PCR; la muestra fue inoculada en diferentes líneas celulares hasta la aparición del efecto citopático. Para confirmar la presencia de SARS-CoV-2 en el cultivo, se utilizó la qRT-PCR a partir de los sobrenadantes, la inmunofluorescencia indirecta (IFI) en células Vero-E6, así como microscopía electrónica y secuenciación de nueva generación (nextgeneration sequencing).Resultados. Se confirmó el aislamiento de SARS-CoV-2 en células Vero-E6 por la aparición del efecto citopático tres días después de la infección, así como mediante la qRT-PCR y la IFI positiva con suero de paciente convaleciente positivo para SARS-CoV-2. Además, en las imágenes de microscopía electrónica de trasmisión y de barrido de células infectadas se observaron estructuras compatibles con viriones de SARS-CoV-2. Por último, se obtuvo la secuencia completa del genoma, lo que permitió clasificar el aislamiento como linaje B.1.5.Conclusiones. La evidencia presentada en este artículo permite confirmar el primer aislamiento de SARS-CoV-2 en Colombia. Además, muestra que esta cepa se comporta en cultivo celular de manera similar a lo reportado en la literatura para otros aislamientos y que su composición genética está acorde con la variante predominante en el mundo. Finalmente, se resalta la importancia que tiene el aislamiento viral para la detección de anticuerpos, para la caracterización genotípica y fenotípica de la cepa y para probar compuestos con potencial antiviral.

Fungal microbiome of barley grain revealed by NGS and mycological analysis

Introduction. Barley can be infected with a broad variety of fungi, which can cause considerable loss of crop yield and reduce the quality of grain. Modern vision on the geographical and ecological distribution and biodiversity of micromycetes has been established by traditional, cultivation-based methods. However, more recently, molecular methods have shifted microbiological research to a new level, making it possible to investigate hidden taxonomical biodiversity. Study objects and methods. For this study, we determined the fungal biome on the surface and inside of barley grains using the traditional mycological method and the contemporary molecular method, which employed DNA metabarcoding based on NGS (nextgeneration sequencing) of the ITS2 region. We analyzed five cultivars that were collected in two subsequent crop seasons (2014, 2015). Results and discussion. DNA metabarcoding revealed 43 operational taxonomic units, while 17 taxa of genus or species level were recovered by the traditional method. DNA metabarcoding revealed several minor species and one predominant, presumably plantpathogenic Phaeosphaeria sp., which were not detected in the agar plate-based assay. Traditionally, Fusarium fungi were identified by mycological assay. However, the resolution of DNA metabarcoding was sufficient to determine main Fusarium groups divided by ability to produce toxic secondary metabolites. The combined list of Ascomycetes consisted of 15 genera, including 14 fungi identified to species level. The list of Basidiomycota derived from DNA metabarcoding data alone included 8 genera. Conclusion. It was found that crop season predetermines the fungal community structure; mycobiota on the surface and inside of grain was significantly different.

The Promise of Whole-exome Sequencing for Prenatal Genetic Diagnosis

Prenatal genetic diagnosis provides information for pregnancy and perinatal decision- making and management. Cytogenetic testing methods, including chromosomal microarray analysis and gene panels, have evolved to become a part of routine laboratory testing, providing valuable diagnostic and prognostic information for prenatal diagnoses. Despite this progress, however, cytogenetic analyses are limited by their resolution and diagnosis is only possible in around 40% of the dysmorphic fetuses. The advent of nextgeneration sequencing (NGS), whole-genome sequencing or whole-exome sequencing has revolutionized prenatal diagnosis and fetal medicine. These technologies have improved the identification of genetic disorders in fetuses with structural abnormalities and provide valuable diagnostic and prognostic information for the detection of genomic defects. Here, the potential future of prenatal genetic diagnosis, including a move toward NGS technologies, is discussed.

Sodium Bisulfite Conversion of Human Genome for DNA Methylation Studies

The regulation of transcription and translation of a gene under a given environment is dependent on several factors and epigenetics is one such factor, responsible for the differential expression of several genes in health and in various diseases. DNA methylation, an important epigenetics mechanism has been shown to play a vital role in numerous cellular processes, and the abnormal patterns of methylation have been linked to the number of human diseases. CpG islands, a short stretch of DNA enriched with CpG sites in the 5’ end of a gene, although remains unmethylated but tends to methylate aberrantly upon certain environmental exposures. The methylation of the promoter region bearing transcriptional start sites of those genes that encodes tumor suppressors such as tumor protein p53, retinoblastoma-associated protein 1, tumor protein p16, breast cancer 1 and many more result in the reduced expression of these genes and have been implicated in a large number of cancers like retinoblastoma, colon, lung and ovarian. A growing number of human diseases have been found to be associated with the aberrant DNA methylation. Hence, a deep insight into the individual’s epigenetic profile is the need of the hour. Several approaches have been developed to map DNA methylation patterns genome-wide. Some of these approaches include enzymatic digestion with methylation-sensitive restriction enzymes, the capture of 5-mC by methylated DNA-binding proteins followed by nextgeneration sequencing and methyl-DNA immunoprecipitation followed by sequencing of precipitated fragments. However, this chapter is going to describe the most recommended method for studying DNA methylation pattern, the method based on bisulfite sequencing. The bisulfite treatment of DNA converts unmethylated cytosine(s) to uracil(s), which are subsequently amplified as Ts by PCR. Hence, the bisulfitetreated DNA has mutations specifically at unmethylated Cs that can be mapped by Next-Generation sequencing.

Clinical and genetic characteristics of new allele variants of the Mowat–Wilson syndrome caused by ZEB2 gene mutations

To date, a large number of monogenic diseases and syndromes, in the clinical picture of which there are convulsions, a psycho-speech development delay and dysmorphic features have been described. One of the hereditary syndromes with a specific phenotype is the Movat–Wilson syndrome. To diagnose the syndrome, a set of survey methods was used: genealogical analysis, neurological examination, evaluation of intellectual development with the help of psychological tests, and sequencing of the new generation exome. As a result of sequencing exome on the panel of genes responsible for the emergence of hereditary epilepsy, two patients of different sex at the age of 10 and 5 years were identified with previously not described mutations in the ZEB2 gene in the heterozygous state. Clinical manifestations of the disease in these patients were of varying degrees of severity, which can be explained in terms of the functional significance of the changes detected. The variety of clinical manifestations of the same disease leads to considerable difficulties in diagnosing, however, due to the introduction of the nextgeneration sequencing in medical practice, the effectiveness of diagnosing hereditary diseases and syndromes, the verification of which has been difficult for a long time, has increased significantly.

Genetics and Hatchery Management: A Parentage-based Tagging Approach to Blueback Herring Conservation

Blueback Herring Alosa aestivalis populations throughout the East Coast have declined precipitously since the late 1980s and were listed as a Species of Concern in 2006 by the National Oceanic and Atmospheric Administration. Natural resource agencies are attempting to restore this species to viable and sustainable levels with fry stockings cultured in hatcheries. To evaluate the long-term contribution of stockings to populations, agencies need an accurate method to track these stocking efforts. Genetic parentage-based tagging is recognized as a feasible means of assessing hatchery contribution of stocked fish to rivers of interest. However, Blueback Herring lack a reliable set of genetic markers to conduct parentage-based tagging. To this end, we analyzed previously described microsatellites as well as new microsatellite markers identified through NextGeneration sequencing to create a suite of 14 Blueback Herring markers useful for parentage-based tagging. The markers were successful in parentage analysis for Blueback Herring collected from the Chowan River, North Carolina. An additional challenge in the management of Blueback Herring is the ability to phenotypically distinguish Blueback Herring from the closely related Alewife Alosa pseudoharengus. Furthermore, recent studies provide evidence that these two species, collectively referred to as river herring, may be hybridizing with one another in some systems. Microsatellite marker AsaC334 can be utilized to discriminate between the two species, as well as to identify their F1 hybrids, thereby providing another genetic tool for hatchery management.

Detection and genome characterization of Potato virus Y isolates infecting potato (Solanum tuberosum L.) in La Union (Antioquia, Colombia)

Potato virus Y (PVY) is one of the most severe viruses affecting the production of potato (Solanum tuberosum) in the world. This study presents a detailed molecular analysis using nextgeneration sequencing (NGS), IC-RT-qPCR and RT-PCR on the PVY isolates infecting seed-tubers and foliage of potato plants cv. Diacol-Capiro in La Union (Antioquia, Colombia). Analysis of incidence by IC-RT-qPCR in 15 random leaf samples of three cultivation plots and fifteen sprouting tuber eye-buds reveal infection levels between 13.4 and 80%; a higher incidence of 86.7% was observed in seed-tuber samples with threshold cycle (Ct) values as low as 24.3. Genome assembly from a bulk of foliage samples resulted in a consensus PVY genome (PVY_LaUnionF) of 9,702 nt and 399 polymorphic sites within the polyprotein ORF; while the assembled genome from sprouts of tubers has 9,704 nt (PVY_LaUnionT) and contained only six polymorphic nucleotide sites. Phylogenetic analysis demonstrates that the PVY isolates from leaf samples are in the recombinant PVYNTN group (sequence identity >99%); while those from tuber sprouts are in the PVYN/NTN group with identities above 95%. Sanger sequencing of viral capsid suggests the presence of a third variant related to PVYO, a prevalent strain reported in potato fields worldwide.

Genotype–phenotype correlation in a cohort of Portuguese patients comprising the entire spectrum of VWD types: impact of NGS

SummaryThe diagnosis of von Willebrand disease (VWD), the most common inherited bleeding disorder, is characterised by a variable bleeding tendency and heterogeneous laboratory phenotype. The sequencing of the entire VWF coding region has not yet become a routine practice in diagnostic laboratories owing to its high costs. Nevertheless, nextgeneration sequencing (NGS) has emerged as an alternative to overcome this limitation. We aimed to determine the correlation of genotype and phenotype in 92 Portuguese individuals from 60 unrelated families with VWD; therefore, we directly sequenced VWF. We compared the classical Sanger sequencing approach and NGS to assess the value-added effect on the analysis of the mutation distribution in different types of VWD. Sixty-two different VWF mutations were identified, 27 of which had not been previously described. NGS detected 26 additional mutations, contributing to a broad overview of the mutant alleles present in each VWD type. Twenty-nine probands (48.3 %) had two or more mutations; in addition, mutations with pleiotropic effects were detected, and NGS allowed an appropriate classification for seven of them. Furthermore, the differential diagnosis between VWD 2B and platelet type VWD (n = 1), Bernard–Soulier syndrome and VWD 2B (n = 1), and mild haemophilia A and VWD 2N (n = 2) was possible. NGS provided an efficient laboratory workflow for analysing VWF. These findings in our cohort of Portuguese patients support the proposal that improving VWD diagnosis strategies will enhance clinical and laboratory approaches, allowing to establish the most appropriate treatment for each patient.