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1.
Discov Med ; 29(157): 129-137, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33002409

RESUMO

Sepsis is a life-threatening clinical condition demanding accurate and rapid diagnosis of the culprit pathogen, thereby to improve prognosis. Pathogen determination through blood culture is the gold standard for diagnosis but has limitations due to low sensitivity. Recently, circulating DNAs derived from pathogenic organisms were found in the plasma of patients with sepsis and were further proved to be more sensitive biomarkers for the diagnosis of the pathogen origin in sepsis. However, the fundamental molecular characteristics of circulating DNA in patients with sepsis remain unclear. Here, we used specific PCR and Sanger sequencing to verify the microbiology culture results via the corresponding plasma circulating DNA. We analyzed the composition and molecular characteristics of circulating DNA in septic patients using next-generation sequencing technology. We showed the presence of pathogen-derived circulating DNA in the plasma of patients with sepsis. The sizes of circulating DNA fragments derived from pathogenic bacteria showed a skewed unimodal distribution, while those derived from host cells showed a normal unimodal distribution. Lengths of fragments at peak concentration for both origins ranged from 150 bp to 200 bp, and reads mapping to pathogenic bacteria genome distributed uniformly on the reference. Our findings have improved our understanding of microbial circulating DNA in patients with sepsis as a potential methodology for the accurate diagnosis of sepsis, especially in light of an urgent need for such a diagnosis associated with the COVID-19 infection.


Assuntos
Infecções Bacterianas/microbiologia , Ácidos Nucleicos Livres/sangue , DNA Bacteriano/sangue , Sepse/microbiologia , Adulto , Idoso , Infecções Bacterianas/complicações , Infecções Bacterianas/diagnóstico , Betacoronavirus , Ácidos Nucleicos Livres/análise , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Técnicas de Cultura , DNA Bacteriano/análise , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/complicações , Pandemias , Pneumonia Viral , Reação em Cadeia da Polimerase , Sepse/complicações , Sepse/diagnóstico , Análise de Sequência de DNA
2.
BMC Bioinformatics ; 21(Suppl 14): 369, 2020 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-32998686

RESUMO

BACKGROUND: Chromosome conformation capture-based methods, especially Hi-C, enable scientists to detect genome-wide chromatin interactions and study the spatial organization of chromatin, which plays important roles in gene expression regulation, DNA replication and repair etc. Thus, developing computational methods to unravel patterns behind the data becomes critical. Existing computational methods focus on intrachromosomal interactions and ignore interchromosomal interactions partly because there is no prior knowledge for interchromosomal interactions and the frequency of interchromosomal interactions is much lower while the search space is much larger. With the development of single-cell technologies, the advent of single-cell Hi-C makes interrogating the spatial structure of chromatin at single-cell resolution possible. It also brings a new type of frequency information, the number of single cells with chromatin interactions between two disjoint chromosome regions. RESULTS: Considering the lack of computational methods on interchromosomal interactions and the unsurprisingly frequent intrachromosomal interactions along the diagonal of a chromatin contact map, we propose a computational method dedicated to analyzing interchromosomal interactions of single-cell Hi-C with this new frequency information. To the best of our knowledge, our proposed tool is the first to identify regions with statistically frequent interchromosomal interactions at single-cell resolution. We demonstrate that the tool utilizing networks and binomial statistical tests can identify interesting structural regions through visualization, comparison and enrichment analysis and it also supports different configurations to provide users with flexibility. CONCLUSIONS: It will be a useful tool for analyzing single-cell Hi-C interchromosomal interactions.


Assuntos
Cromossomos/metabolismo , Análise de Célula Única/métodos , Animais , Cromatina/metabolismo , Fase G1 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Fase S , Zigoto/citologia , Zigoto/metabolismo
3.
BMC Infect Dis ; 20(1): 721, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004020

RESUMO

BACKGROUND: Listeria monocytogenes (L. monocytogenes) is a facultative intracellular bacterial pathogen which can invade different mammalian cells and reach to the central nervous system (CNS), leading to meningoencephalitis and brain abscesses. In the diagnosis of L. monocytogenes meningoencephalitis (LMM), the traditional test often reports negative owing to the antibiotic treatment or a low number of bacteria in the cerebrospinal fluid. To date, timely diagnosis and accurate treatment remains a challenge for patients with listeria infections. CASE PRESENTATION: We present the case of a 66-year-old woman whose clinical manifestations were suspected as tuberculous meningoencephalitis, but the case was finally properly diagnosed as LMM by next-generation sequencing (NGS). The patient was successfully treated using a combined antibacterial therapy, comprising ampicillin and trimethoprim-sulfamethoxazole. CONCLUSION: To improve the sensitivity of LMM diagnosis, we used NGS for the detection of L. monocytogenes. Hence, the clinical utility of this approach can be very helpful since it provides quickly and trust results.


Assuntos
Listeria monocytogenes/genética , Meningite por Listeria/microbiologia , Meningoencefalite/microbiologia , Idoso , Ampicilina/uso terapêutico , Antibacterianos/uso terapêutico , Abscesso Encefálico/tratamento farmacológico , Erros de Diagnóstico , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Listeria monocytogenes/isolamento & purificação , Meningite por Listeria/diagnóstico , Meningite por Listeria/tratamento farmacológico , Meningoencefalite/diagnóstico , Meningoencefalite/tratamento farmacológico , Combinação Trimetoprima e Sulfametoxazol/uso terapêutico , Tuberculose Meníngea/diagnóstico , Tuberculose Meníngea/microbiologia
5.
Medicine (Baltimore) ; 99(41): e22615, 2020 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-33031319

RESUMO

RATIONALE: Limb-girdle muscular dystrophy (LGMD) is a genetic disease, which is characterized by muscle atrophy and weakness mainly involving proximal muscles. Accurate diagnosis of LGMD patient is very important for the appropriate management and long-term prognosis. PATIENT CONCERNS: An 18-year-old woman presented with progressive weakness of limbs, persistent elevated serum creatine kinase, myogenic damages in electromyography, and dysferlin protein deficiency in muscle biopsy. Further next-generation sequencing (NGS) revealed a compound heterozygous variant in dysferlin gene (DYSF), including a novel frameshift variant of c.4010delT. DIAGNOSIS: The patient was diagnosed with LGMD2B clinically and genetically. INTERVENTIONS: Oral levocarnitine and coenzyme Q10 were prescribed to the patient. OUTCOMES: After symptomatic treatments for 1 week, the patient's symptoms were not improved. LESSONS: NGS might be a helpful tool for the diagnosis of LGMD. A novel variant of c.4010delT in DYSF was identified in this case, which broadens the genetic spectrum of LGMD2B.


Assuntos
Disferlina/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Adolescente , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos
6.
Nat Commun ; 11(1): 4932, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004838

RESUMO

Most genes associated with neurodevelopmental disorders (NDDs) were identified with an excess of de novo mutations (DNMs) but the significance in case-control mutation burden analysis is unestablished. Here, we sequence 63 genes in 16,294 NDD cases and an additional 62 genes in 6,211 NDD cases. By combining these with published data, we assess a total of 125 genes in over 16,000 NDD cases and compare the mutation burden to nonpsychiatric controls from ExAC. We identify 48 genes (25 newly reported) showing significant burden of ultra-rare (MAF < 0.01%) gene-disruptive mutations (FDR 5%), six of which reach family-wise error rate (FWER) significance (p < 1.25E-06). Among these 125 targeted genes, we also reevaluate DNM excess in 17,426 NDD trios with 6,499 new autism trios. We identify 90 genes enriched for DNMs (FDR 5%; e.g., GABRG2 and UIMC1); of which, 61 reach FWER significance (p < 3.64E-07; e.g., CASZ1). In addition to doubling the number of patients for many NDD risk genes, we present phenotype-genotype correlations for seven risk genes (CTCF, HNRNPU, KCNQ3, ZBTB18, TCF12, SPEN, and LEO1) based on this large-scale targeted sequencing effort.


Assuntos
Predisposição Genética para Doença , Transtornos do Neurodesenvolvimento/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fator de Ligação a CCCTC/genética , Estudos de Casos e Controles , Estudos de Coortes , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Feminino , Estudos de Associação Genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Canal de Potássio KCNQ3/genética , Masculino , Mutação , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética
7.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 45(9): 1035-1043, 2020.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-33051416

RESUMO

OBJECTIVES: Long non-coding RNAs (lncRNAs) were involved in the development and regulation of necrotizing enterocolitis (NEC) in premature infants. To investigate the changes of lncRNA expression profile in intestinal tissues of NEC for its possible mechanisms. METHODS: Intestinal samples were collected from 11 patients with NEC who needed surgery(the NEC group), and 7 from neonatal non-NEC patients with surgery (the Control group).LncRNA's changes in intestinal samples (3 in the Control group and 3 in the NEC group) were analyzed with high-throughput sequencing.Part of the remaining samples were detected by real-time polymerase chain reaction (real-time PCR), and the results were used to validate the results of high-throughput sequencing. Gene Ontology (GO) analysis and KEGG signaling pathway analysis were performed on differentially expressed genes. RESULTS: There were 5 257 different lncRNAs between the control group and the NEC group. The results of up-regulated lncRNAs (NONHSAG008675.3, NONHSAG020715.2, NONHSAG038187.2) and down-regulated lncRNA (NONHSAG028744.3) were confirmed to be consistent with the results of high-throughput sequencing. Expressions of DUOX2, IL-6, TNF, and SAA1 were up-regulated in intestinal tissues of NEC. GO analysis showed that the different lncRNAs were involved in regulation of stimulation, molecular junction and function, and signal transduction and transcription. KEGG analysis identified mainly biological pathways involved in inflammatory bowel disease, PI3K-Akt, NF-κB, etc. CONCLUSIONS: LncRNAs might be involved in the pathogenesis of NEC and the inflammation-related lncRNAs may be one of the key factors.


Assuntos
Enterocolite Necrosante , RNA Longo não Codificante , Animais , Oxidases Duais , Enterocolite Necrosante/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Intestinos , Fosfatidilinositol 3-Quinases , RNA Longo não Codificante/genética
8.
Int J Med Sci ; 17(16): 2449-2453, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33029087

RESUMO

The COVID-19 pandemic is a novel infectious disease pandemic with the agent SARS-CoV-2 virus which is currently affecting and causing damage globally. The outbreak has been crossing over 200 countries in the world. In the situation of the outbreak of COVID-19, Vietnam has first sixteen typical cases confirmed positive updated to Feb 28th, 2020. After completely applying the medical prevention and active control, Vietnam has the ability to take control of the outbreak of COVID-19 as a recent of WHO assessment. Vietnam has been reported as an effective country for prevention and control the outbreak of COVID-19. We retroactive reviewed our experience with 16 positive cases isolation. This article aims to present the first cohort of COVID-19 patients updated to Feb 28th, 2020 in Vietnam and sharing the national response to the pandemic.


Assuntos
Betacoronavirus , Técnicas de Laboratório Clínico , Infecções por Coronavirus/tratamento farmacológico , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Betacoronavirus/genética , Estudos de Coortes , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/etiologia , Infecções por Coronavirus/transmissão , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Programas Nacionais de Saúde , Pneumonia Viral/etiologia , Pneumonia Viral/transmissão , Vietnã/epidemiologia
9.
JAMA Netw Open ; 3(10): e2024191, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33026453

RESUMO

Importance: In late December 2019, an outbreak of a novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. Data on the routes of transmission to Los Angeles, California, the US West Coast epicenter for coronavirus disease 2019 (COVID-19), and subsequent community spread are limited. Objective: To determine the transmission routes of SARS-CoV-2 to Southern California and elucidate local community spread within the Los Angeles metropolitan area. Design, Setting, and Participants: This case series included 192 consecutive patients with reverse transcription-polymerase chain reaction (RT-PCR) test results positive for SARS-CoV-2 who were evaluated at Cedars-Sinai Medical Center in Los Angeles, California, from March 22 to April 15, 2020. Data analysis was performed from April to May 2020. Main Outcomes and Measures: SARS-CoV-2 viral genomes were sequenced. Los Angeles isolates were compared with genomes from global subsampling and from New York, New York; Washington state; and China to determine potential sources of viral dissemination. Demographic data and outcomes were collected. Results: The cohort included 192 patients (median [interquartile range] age, 59.5 [43-75] years; 110 [57.3%] men). The genetic characterization of SARS-CoV-2 isolates in the Los Angeles population pinpointed community transmission of 13 patients within a 3.81 km2 radius. Variation landscapes of this case series also revealed a cluster of 10 patients that contained 5 residents at a skilled nursing facility, 1 resident of a nearby skilled nursing facility, 3 health care workers, and a family member of a resident of one of the skilled nursing facilities. Person-to-person transmission was detected in a cluster of 5 patients who shared the same single-nucleotide variation in their SARS-CoV-2 genomes. High viral genomic diversity was identified: 20 Los Angeles isolates (15.0%) resembled SARS-CoV-2 genomes from Asia, while 109 Los Angeles isolates (82.0%) were similar to isolates originating from Europe. Analysis of other common respiratory viral pathogens did not reveal coinfection in the cohort. Conclusions and Relevance: These findings highlight the precision of detecting person-to-person transmission and accurate contact tracing directly through SARS-CoV-2 genome isolation and sequencing. Development and application of phylogenetic analyses from the Los Angeles population established connections between COVID-19 clusters locally and throughout the US.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/transmissão , Genoma Viral/genética , Pneumonia Viral/transmissão , Adulto , Idoso , Ásia , California/epidemiologia , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , Europa (Continente) , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Los Angeles/epidemiologia , Masculino , Pessoa de Meia-Idade , Cidade de Nova Iorque , Pandemias , Filogenia , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , Análise de Sequência de RNA , Proteínas não Estruturais Virais/genética , Washington
10.
Medicine (Baltimore) ; 99(35): e21938, 2020 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-32871937

RESUMO

RATIONALE: Aggressive variant of splenic marginal zone lymphoma (AV-SMZL) is a very rare disease that is often associated with TP53 mutations and has a poor prognosis. On the other hand, recent advances in genome sequencing techniques enable us to understand the molecular characteristics of rare cancers such as AV-SMZL. Here we present a case of AV-SMZL analyzed using a genetic test. PATIENT CONCERNS: A 66-year-old woman was admitted with splenomegaly and lymphocytosis. Computed tomography revealed marked splenomegaly without lymphadenopathy in any other areas. The serum soluble interleukin-2 receptor (sIL-2R) level was significantly elevated. Peripheral and bone marrow blood tests showed an increase in abnormal lymphocytes. DIAGNOSIS: A splenectomy revealed an SMZL pattern with increased numbers of large cells and mitotic cells and a high Ki-67 positivity rate, which led to a diagnosis of AV-SMZL. Although TP53 mutation was not detected, mutations in NOTCH2, NCOA4, PTEN, EPHA3, and KMT2D were identified. Among these, the mutations in NCOA4, PTEN, and EPHA3 were novel pathogenic mutations in SMZL, which suggests they may be related to the aggressiveness and persistence of the disease. INTERVENTIONS: The patient was administered a rituximab-containing regimen and rituximab-maintenance therapy. OUTCOMES: The patient continues to exhibit a complete response. LESSONS: This is a case of AV-SMZL in which a cancer panel test successfully detected genetic alterations that are potentially associated with its pathogenesis. These findings suggest that genetic analysis is useful for making diagnoses as well as for determining treatment strategies in AV-SMZL.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfoma de Zona Marginal Tipo Células B/diagnóstico , Linfoma de Zona Marginal Tipo Células B/tratamento farmacológico , Rituximab/uso terapêutico , Neoplasias Esplênicas/diagnóstico , Neoplasias Esplênicas/tratamento farmacológico , Idoso , Análise Mutacional de DNA , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma de Zona Marginal Tipo Células B/cirurgia , Mutação , Indução de Remissão , Esplenectomia , Neoplasias Esplênicas/genética , Neoplasias Esplênicas/cirurgia
11.
BMC Infect Dis ; 20(1): 648, 2020 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-32883215

RESUMO

BACKGROUND: Due to the frequent reassortment and zoonotic potential of influenza A viruses, rapid gain of sequence information is crucial. Alongside established next-generation sequencing protocols, the MinION sequencing device (Oxford Nanopore Technologies) has become a serious competitor for routine whole-genome sequencing. Here, we established a novel, rapid and high-throughput MinION multiplexing workflow based on a universal RT-PCR. METHODS: Twelve representative influenza A virus samples of multiple subtypes were universally amplified in a one-step RT-PCR and subsequently sequenced on the MinION instrument in conjunction with a barcoding library preparation kit from the rapid family and the MinIT performing live base-calling. The identical PCR products were sequenced on an IonTorrent platform and, after final consensus assembly, all data was compared for validation. To prove the practicability of the MinION-MinIT method in human and veterinary diagnostics, we sequenced recent and historical influenza strains for further benchmarking. RESULTS: The MinION-MinIT combination generated over two million reads for twelve samples in a six-hour sequencing run, from which a total of 72% classified as quality screened, trimmed and mapped influenza reads to produce full genome sequences. Identities between the datasets of > 99.9% were achieved, with 100% coverage of all segments alongside a sufficient confidence and 4492fold mean depth. From RNA extraction to finished sequences, only 14 h were required. CONCLUSIONS: Overall, we developed and validated a novel and rapid multiplex workflow for influenza A virus sequencing. This protocol suits both clinical and academic settings, aiding in real time diagnostics and passive surveillance.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Vírus da Influenza A/genética , Sequenciamento por Nanoporos/métodos , Humanos , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Fluxo de Trabalho
12.
J Clin Virol ; 131: 104594, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32866812

RESUMO

INTRODUCTION: The SARS-CoV-2 pandemic of 2020 is a prime example of the omnipresent threat of emerging viruses that can infect humans. A protocol for the identification of novel coronaviruses by viral metagenomic sequencing in diagnostic laboratories may contribute to pandemic preparedness. AIM: The aim of this study is to validate a metagenomic virus discovery protocol as a tool for coronavirus pandemic preparedness. METHODS: The performance of a viral metagenomic protocol in a clinical setting for the identification of novel coronaviruses was tested using clinical samples containing SARS-CoV-2, SARS-CoV, and MERS-CoV, in combination with databases generated to contain only viruses of before the discovery dates of these coronaviruses, to mimic virus discovery. RESULTS: Classification of NGS reads using Centrifuge and Genome Detective resulted in assignment of the reads to the closest relatives of the emerging coronaviruses. Low nucleotide and amino acid identity (81% and 84%, respectively, for SARS-CoV-2) in combination with up to 98% genome coverage were indicative for a related, novel coronavirus. Capture probes targeting vertebrate viruses, designed in 2015, enhanced both sequencing depth and coverage of the SARS-CoV-2 genome, the latter increasing from 71% to 98%. CONCLUSION: The model used for simulation of virus discovery enabled validation of the metagenomic sequencing protocol. The metagenomic protocol with virus probes designed before the pandemic, can assist the detection and identification of novel coronaviruses directly in clinical samples.


Assuntos
Infecções por Coronavirus/virologia , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala , Metagenômica , Pneumonia Viral/virologia , Betacoronavirus/isolamento & purificação , Técnicas de Laboratório Clínico/métodos , Biologia Computacional , Infecções por Coronavirus/diagnóstico , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Nasofaringe/virologia , Pandemias , Vírus da SARS/isolamento & purificação
13.
Ann Saudi Med ; 40(5): 373-381, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32954791

RESUMO

BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. OBJECTIVES: Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay. MATERIALS AND METHODS: Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes). RESULTS: Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media. CONCLUSIONS: We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing. LIMITATIONS: Small patient sample size. CONFLICT OF INTEREST: None.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Nasofaringe/virologia , Pneumonia Viral/diagnóstico , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Animais , Automação , Chlorocebus aethiops , Técnicas de Laboratório Clínico , Vírus da Encefalomiocardite/genética , Humanos , Levivirus/genética , Proteínas do Nucleocapsídeo/genética , Pandemias , RNA Replicase/genética , RNA Viral/análise , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/genética , Células Vero , Proteínas do Envelope Viral/genética , Proteínas não Estruturais Virais/genética
14.
PLoS Comput Biol ; 16(9): e1008173, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946435

RESUMO

Single-cell Hi-C (scHi-C) interrogates genome-wide chromatin interaction in individual cells, allowing us to gain insights into 3D genome organization. However, the extremely sparse nature of scHi-C data poses a significant barrier to analysis, limiting our ability to tease out hidden biological information. In this work, we approach this problem by applying topic modeling to scHi-C data. Topic modeling is well-suited for discovering latent topics in a collection of discrete data. For our analysis, we generate nine different single-cell combinatorial indexed Hi-C (sci-Hi-C) libraries from five human cell lines (GM12878, H1Esc, HFF, IMR90, and HAP1), consisting over 19,000 cells. We demonstrate that topic modeling is able to successfully capture cell type differences from sci-Hi-C data in the form of "chromatin topics." We further show enrichment of particular compartment structures associated with locus pairs in these topics.


Assuntos
Cromatina , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Célula Única/métodos , Linhagem Celular , Cromatina/química , Cromatina/genética , Análise por Conglomerados , Biblioteca Gênica , Humanos , Processamento de Linguagem Natural
15.
Nature ; 585(7825): 459-463, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908305

RESUMO

The RNA polymerase II (Pol II) core promoter is the strategic site of convergence of the signals that lead to the initiation of DNA transcription1-5, but the downstream core promoter in humans has been difficult to understand1-3. Here we analyse the human Pol II core promoter and use machine learning to generate predictive models for the downstream core promoter region (DPR) and the TATA box. We developed a method termed HARPE (high-throughput analysis of randomized promoter elements) to create hundreds of thousands of DPR (or TATA box) variants, each with known transcriptional strength. We then analysed the HARPE data by support vector regression (SVR) to provide comprehensive models for the sequence motifs, and found that the SVR-based approach is more effective than a consensus-based method for predicting transcriptional activity. These results show that the DPR is a functionally important core promoter element that is widely used in human promoters. Notably, there appears to be a duality between the DPR and the TATA box, as many promoters contain one or the other element. More broadly, these findings show that functional DNA motifs can be identified by machine learning analysis of a comprehensive set of sequence variants.


Assuntos
Sequência Consenso/genética , Regulação da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Máquina de Vetores de Suporte , Transcrição Genética , Sequência de Bases , Células/metabolismo , Simulação por Computador , Conjuntos de Dados como Assunto , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Genéticos , Mutagênese , TATA Box/genética
16.
BMC Bioinformatics ; 21(Suppl 8): 299, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938362

RESUMO

BACKGROUND: The development of Next Generation Sequencing (NGS) has had a major impact on the study of genetic sequences. Among problems that researchers in the field have to face, one of the most challenging is the taxonomic classification of metagenomic reads, i.e., identifying the microorganisms that are present in a sample collected directly from the environment. The analysis of environmental samples (metagenomes) are particularly important to figure out the microbial composition of different ecosystems and it is used in a wide variety of fields: for instance, metagenomic studies in agriculture can help understanding the interactions between plants and microbes, or in ecology, they can provide valuable insights into the functions of environmental communities. RESULTS: In this paper, we describe a new lightweight alignment-free and assembly-free framework for metagenomic classification that compares each unknown sequence in the sample to a collection of known genomes. We take advantage of the combinatorial properties of an extension of the Burrows-Wheeler transform, and we sequentially scan the required data structures, so that we can analyze unknown sequences of large collections using little internal memory. The tool LiME (Lightweight Metagenomics via eBWT) is available at https://github.com/veronicaguerrini/LiME . CONCLUSIONS: In order to assess the reliability of our approach, we run several experiments on NGS data from two simulated metagenomes among those provided in benchmarking analysis and on a real metagenome from the Human Microbiome Project. The experiment results on the simulated data show that LiME is competitive with the widely used taxonomic classifiers. It achieves high levels of precision and specificity - e.g. 99.9% of the positive control reads are correctly assigned and the percentage of classified reads of the negative control is less than 0.01% - while keeping a high sensitivity. On the real metagenome, we show that LiME is able to deliver classification results comparable to that of MagicBlast. Overall, the experiments confirm the effectiveness of our method and its high accuracy even in negative control samples.


Assuntos
Algoritmos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metagenômica/métodos , Humanos , Reprodutibilidade dos Testes
17.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 45(7): 849-855, 2020 Jul 28.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-32879089

RESUMO

The 16S rRNA gene is the most commonly used molecular marker for identifying microorganisms. It is used in sequencing technology, including the first-generation, the second-generation, and the third-generation sequencing technology. A large number of studies on the 16S rRNA gene have contributed to a deeper understanding of oral microbial diversity. In the healthy oral cavity, there is microbial diversity in time and space. With the occurrence or development of oral diseases such as caries, periodontal disease, or halitosis, the microbial diversity will be changed.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Boca , RNA Ribossômico 16S/genética
20.
BMC Bioinformatics ; 21(1): 397, 2020 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-32907531

RESUMO

BACKGROUND: Ion Torrent is one of the major next generation sequencing (NGS) technologies and it is frequently used in medical research and diagnosis. The built-in software for the Ion Torrent sequencing machines delivers the sequencing results in the BAM format. In addition to the usual SAM/BAM fields, the Ion Torrent BAM file includes technology-specific flow signal data. The flow signals occupy a big portion of the BAM file (about 75% for the human genome). Compressing SAM/BAM into CRAM format significantly reduces the space needed to store the NGS results. However, the tools for generating the CRAM formats are not designed to handle the flow signals. This missing feature has motivated us to develop a new program to improve the compression of the Ion Torrent files for long term archiving. RESULTS: In this paper, we present IonCRAM, the first reference-based compression tool to compress Ion Torrent BAM files for long term archiving. For the BAM files, IonCRAM could achieve a space saving of about 43%. This space saving is superior to what achieved with the CRAM format by about 8-9%. CONCLUSIONS: Reducing the space consumption of NGS data reduces the cost of storage and data transfer. Therefore, developing efficient compression software for clinical NGS data goes beyond the computational interest; as it ultimately contributes to the overall cost reduction of the clinical test. The space saving achieved by our tool is a practical step in this direction. The tool is open source and available at Code Ocean, github, and http://ioncram.saudigenomeproject.com .


Assuntos
Interface Usuário-Computador , Algoritmos , Bases de Dados Genéticas , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos
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