Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 34.169
Filtrar
1.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34065034

RESUMO

Seed germination is a key step in the new life cycle of plants. In agriculture, we regard the rapid and consistent process of seed germination as one of the necessary conditions to measure the high quality and yield of crops. ENO2 is a key enzyme in glycolysis, which also plays an important role in plant growth and abiotic stress responses. In our study, we found that the time of seed germination in AtENO2 mutation (eno2-) was earlier than that of wild type (WT) in Arabidopsis thaliana. Previous studies have shown that microRNAs (miRNAs) were vital in seed germination. After deep sequencing of small RNA, we found 590 differentially expressed miRNAs in total, of which 87 were significantly differentially expressed miRNAs. By predicting the target genes of miRNAs and analyzing the GO annotation, we have counted 18 genes related to seed germination, including ARF family, TIR1, INVC, RR19, TUDOR2, GA3OX2, PXMT1, and TGA1. MiR9736-z, miR5059-z, ath-miR167a-5p, ath-miR167b, ath-miR5665, ath-miR866-3p, miR10186-z, miR8165-z, ath-miR857, ath-miR399b, ath-miR399c-3p, miR399-y, miR163-z, ath-miR393a-5p, and ath-miR393b-5p are the key miRNAs regulating seed germination-related genes. Through KEGG enrichment analysis, we found that phytohormone signal transduction pathways were significantly enriched, and these miRNAs mentioned above also participate in the regulation of the genes in plant hormone signal transduction pathways, thus affecting the synthesis of plant hormones and further affecting the process of seed germination. This study laid the foundation for further exploration of the AtENO2 regulation for seed germination.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Redes Reguladoras de Genes , Germinação , RNA de Plantas/genética , Pequeno RNA não Traduzido/genética , Sementes/fisiologia , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/genética , Sementes/genética
2.
BMC Med Genomics ; 14(1): 144, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34074255

RESUMO

BACKGROUND: Transmission within families and multiple spike protein mutations have been associated with the rapid transmission of SARS-CoV-2. We aimed to: (1) describe full genome characterization of SARS-CoV-2 and correlate the sequences with epidemiological data within family clusters, and (2) conduct phylogenetic analysis of all samples from Yogyakarta and Central Java, Indonesia and other countries. METHODS: The study involved 17 patients with COVID-19, including two family clusters. We determined the full-genome sequences of SARS-CoV-2 using the Illumina MiSeq next-generation sequencer. Phylogenetic analysis was performed using a dataset of 142 full-genomes of SARS-CoV-2 from different regions. RESULTS: Ninety-four SNPs were detected throughout the open reading frame (ORF) of SARS-CoV-2 samples with 58% (54/94) of the nucleic acid changes resulting in amino acid mutations. About 94% (16/17) of the virus samples showed D614G on spike protein and 56% of these (9/16) showed other various amino acid mutations on this protein, including L5F, V83L, V213A, W258R, Q677H, and N811I. The virus samples from family cluster-1 (n = 3) belong to the same clade GH, in which two were collected from deceased patients, and the other from the survived patient. All samples from this family cluster revealed a combination of spike protein mutations of D614G and V213A. Virus samples from family cluster-2 (n = 3) also belonged to the clade GH and showed other spike protein mutations of L5F alongside the D614G mutation. CONCLUSIONS: Our study is the first comprehensive report associating the full-genome sequences of SARS-CoV-2 with the epidemiological data within family clusters. Phylogenetic analysis revealed that the three viruses from family cluster-1 formed a monophyletic group, whereas viruses from family cluster-2 formed a polyphyletic group indicating there is the possibility of different sources of infection. This study highlights how the same spike protein mutations among members of the same family might show different disease outcomes.


Assuntos
COVID-19/epidemiologia , RNA Viral/genética , SARS-CoV-2/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , COVID-19/patologia , COVID-19/virologia , Criança , Família , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Indonésia/epidemiologia , Masculino , Pessoa de Meia-Idade , Mutação , Filogenia , RNA Viral/química , SARS-CoV-2/classificação , SARS-CoV-2/isolamento & purificação , Índice de Gravidade de Doença , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Sequenciamento Completo do Genoma
3.
BMC Bioinformatics ; 22(1): 304, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34090332

RESUMO

BACKGROUND: The detection of genome variants, including point mutations, indels and structural variants, is a fundamental and challenging computational problem. We address here the problem of variant detection between two deep-sequencing (DNA-seq) samples, such as two human samples from an individual patient, or two samples from distinct bacterial strains. The preferred strategy in such a case is to align each sample to a common reference genome, collect all variants and compare these variants between samples. Such mapping-based protocols have several limitations. DNA sequences with large indels, aggregated mutations and structural variants are hard to map to the reference. Furthermore, DNA sequences cannot be mapped reliably to genomic low complexity regions and repeats. RESULTS: We introduce 2-kupl, a k-mer based, mapping-free protocol to detect variants between two DNA-seq samples. On simulated and actual data, 2-kupl achieves higher accuracy than other mapping-free protocols. Applying 2-kupl to prostate cancer whole exome sequencing data, we identify a number of candidate variants in hard-to-map regions and propose potential novel recurrent variants in this disease. CONCLUSIONS: We developed a mapping-free protocol for variant calling between matched DNA-seq samples. Our protocol is suitable for variant detection in unmappable genome regions or in the absence of a reference genome.


Assuntos
Genômica , Sequenciamento de Nucleotídeos em Larga Escala , DNA , Genoma Humano , Humanos , Análise de Sequência de DNA
4.
BMC Bioinformatics ; 22(1): 303, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34090340

RESUMO

BACKGROUND: Long-read sequencing is revolutionizing genome assembly: as PacBio and Nanopore technologies become more accessible in technicity and in cost, long-read assemblers flourish and are starting to deliver chromosome-level assemblies. However, these long reads are usually error-prone, making the generation of a haploid reference out of a diploid genome a difficult enterprise. Failure to properly collapse haplotypes results in fragmented and structurally incorrect assemblies and wreaks havoc on orthology inference pipelines, yet this serious issue is rarely acknowledged and dealt with in genomic projects, and an independent, comparative benchmark of the capacity of assemblers and post-processing tools to properly collapse or purge haplotypes is still lacking. RESULTS: We tested different assembly strategies on the genome of the rotifer Adineta vaga, a non-model organism for which high coverages of both PacBio and Nanopore reads were available. The assemblers we tested (Canu, Flye, NextDenovo, Ra, Raven, Shasta and wtdbg2) exhibited strikingly different behaviors when dealing with highly heterozygous regions, resulting in variable amounts of uncollapsed haplotypes. Filtering reads generally improved haploid assemblies, and we also benchmarked three post-processing tools aimed at detecting and purging uncollapsed haplotypes in long-read assemblies: HaploMerger2, purge_haplotigs and purge_dups. CONCLUSIONS: We provide a thorough evaluation of popular assemblers on a non-model eukaryote genome with variable levels of heterozygosity. Our study highlights several strategies using pre and post-processing approaches to generate haploid assemblies with high continuity and completeness. This benchmark will help users to improve haploid assemblies of non-model organisms, and evaluate the quality of their own assemblies.


Assuntos
Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Genoma , Haplótipos , Análise de Sequência de DNA
5.
BMC Ophthalmol ; 21(1): 249, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34090370

RESUMO

BACKGROUND: ALG3-CDG is a rare autosomal recessive disease. It is characterized by deficiency of alpha-1,3-mannosyltransferase caused by pathogenic variants in the ALG3 gene. Patients manifest with severe neurologic, cardiac, musculoskeletal and ophthalmic phenotype in combination with dysmorphic features, and almost half of them die before or during the neonatal period. CASE PRESENTATION: A 23 months-old girl presented with severe developmental delay, epilepsy, cortical atrophy, cerebellar vermis hypoplasia and ocular impairment. Facial dysmorphism, clubfeet and multiple joint contractures were observed already at birth. Transferrin isoelectric focusing revealed a type 1 pattern. Funduscopy showed hypopigmentation and optic disc pallor. Profound retinal ganglion cell loss and inner retinal layer thinning was documented on spectral-domain optical coherence tomography imaging. The presence of optic nerve hypoplasia was also supported by magnetic resonance imaging. A gene panel based next-generation sequencing and subsequent Sanger sequencing identified compound heterozygosity for two novel variants c.116del p.(Pro39Argfs*40) and c.1060 C > T p.(Arg354Cys) in ALG3. CONCLUSIONS: Our study expands the spectrum of pathogenic variants identified in ALG3. Thirty-three variants in 43 subjects with ALG3-CDG have been reported. Literature review shows that visual impairment in ALG3-CDG is most commonly linked to optic nerve hypoplasia.


Assuntos
Defeitos Congênitos da Glicosilação , Degeneração Retiniana , Pré-Escolar , Defeitos Congênitos da Glicosilação/genética , Olho , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Manosiltransferases/genética , Fenótipo
6.
J Transl Med ; 19(1): 246, 2021 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-34090468

RESUMO

BACKGROUND: Since the first complete genome sequencing of SARS-CoV-2 in December 2019, more than 550,000 genomes have been submitted into the GISAID database. Sequencing of the SARS-CoV-2 genome might allow identification of variants with increased contagiousness, different clinical patterns and/or different response to vaccines. A highly automated next generation sequencing (NGS)-based method might facilitate an active genomic surveillance of the virus. METHODS: RNA was extracted from 27 nasopharyngeal swabs obtained from citizens of the Italian Campania region in March-April 2020 who tested positive for SARS-CoV-2. Following viral RNA quantification, sequencing was performed using the Ion AmpliSeq SARS-CoV-2 Research Panel on the Genexus Integrated Sequencer, an automated technology for library preparation and sequencing. The SARS-CoV-2 complete genomes were built using the pipeline SARS-CoV-2 RECoVERY (REconstruction of COronaVirus gEnomes & Rapid analYsis) and analysed by IQ-TREE software. RESULTS: The complete genome (100%) of SARS-CoV-2 was successfully obtained for 21/27 samples. In particular, the complete genome was fully sequenced for all 15 samples with high viral titer (> 200 copies/µl), for the two samples with a viral genome copy number < 200 but greater than 20, and for 4/10 samples with a viral load < 20 viral copies. The complete genome sequences classified into the B.1 and B.1.1 SARS-CoV-2 lineages. In comparison to the reference strain Wuhan-Hu-1, 48 total nucleotide variants were observed with 26 non-synonymous substitutions, 18 synonymous and 4 reported in untranslated regions (UTRs). Ten of the 26 non-synonymous variants were observed in ORF1ab, 7 in S, 1 in ORF3a, 2 in M and 6 in N genes. CONCLUSIONS: The Genexus system resulted successful for SARS-CoV-2 complete genome sequencing, also in cases with low viral copies. The use of this highly automated system might facilitate the standardization of SARS-CoV-2 sequencing protocols and make faster the identification of novel variants during the pandemic.


Assuntos
COVID-19 , SARS-CoV-2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Itália , Sequenciamento Completo do Genoma
7.
No Shinkei Geka ; 49(3): 520-526, 2021 May.
Artigo em Japonês | MEDLINE | ID: mdl-34092557

RESUMO

In Japan, two types of cancer gene panel tests have been covered by insurance since 2019, marking the start of the "first year of cancer genomic medicine". Cancer genomic medicine is a medical treatment in which a large number of genes are analyzed at once by next generation sequencing(NGS), mainly using cancer tissues, to reveal the genetic alterations in the patient's cancer so that the best treatment can be applied for each patient. In order to present actual treatment based on the results of the cancer gene panel test, it is necessary to prepare a report by an "expert panel", which is a group of multidisciplinary experts. Although the current percentage of patients who actually receive treatment is approximately 13% in total, it is expected to improv in the future. The number of clinical trials where brain tumor patients can participate is still small, but a certain number of patients are actually benefiting from these new drugs. We hoped that cancer genomic medicine for brain tumor patients will be actively promoted in the future.


Assuntos
Neoplasias Encefálicas , Genômica , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Japão , Mutação
8.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(6): 513-520, 2021 Jun 10.
Artigo em Chinês | MEDLINE | ID: mdl-34096016

RESUMO

The use of whole exome sequencing (WES) for the detection of disease-causing variants of genetic diseases and for non-invasive prenatal screening (NIPS) of fetal aneuploidies are two major clinical applications of next generation sequencing (NGS). This article has summarized the official documents developed and updated by the American College of Medical Genetics and Genomics (ACMG) on governing WES and NIPS. These include the development of expert consensus policies and position statements on an ongoing basis to guide clinical application of NGS technology and variant analysis, establish evidence-based practical resources, as well as standards and guidelines to govern diagnosis and screening. These ACMG documents are valuable references to Chinese geneticists, but direct adoption of these standards and guidelines may not be practical due to the differences in disease-associated variant frequencies in Chinese population, socioeconomic status, and medical practice between the two countries. It is hoped that this review could facilitate the development of NGS and NIPS standards and guidelines that are consistent with international standards and concordant with medical genetics practice in China to provide high-quality, efficient and safe clinical services for patients and their families with genetic diseases.


Assuntos
Genômica , Sequenciamento de Nucleotídeos em Larga Escala , China , Consenso , Feminino , Humanos , Gravidez , Tecnologia , Estados Unidos
9.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 38(6): 545-548, 2021 Jun 10.
Artigo em Chinês | MEDLINE | ID: mdl-34096022

RESUMO

OBJECTIVE: To explore the genetic basis of a pedigree affected with Alagille syndrome (ALGS). METHODS: Targeted capture and next generation sequencing was carried out for the proband. Candidate variants were verified by Sanger sequencing among his family members. Their pathogenicity of the variant was predicted with bioinformatic analysis. Clinical characteristics and genotype-phenotype correlation were analyzed. RESULTS: The proband, his elder sister and mother were found to carry a heterozygous c.1270dupG (p.Ala424Glyfs*5) variant of the JAG1 gene, which may lead to premature termination of translation and a truncated protein with loss of function. The variant was unreported previously. The phenotypes of the proband (cholestasis, pulmonary artery stenosis and peculiar faces) have differed from those of his elder sister (cholestasis with pruritus, posterior embryonic ring of cornea) and mother (with no clinical manifestation). Cholestasis and peculiar face of the proband became insignificant with age. CONCLUSION: The c.1270dupG (p.Ala424Glyfs*5) variant of the JAG1 gene probably underlay the ALGS in this pedigree with incomplete penetrance.


Assuntos
Síndrome de Alagille , Idoso , Síndrome de Alagille/genética , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Linhagem , Fenótipo
10.
Viruses ; 13(6)2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071591

RESUMO

Several recently developed high-throughput techniques have changed the field of molecular virology. For example, proteomics studies reveal complete interactomes of a viral protein, genome-wide CRISPR knockout and activation screens probe the importance of every single human gene in aiding or fighting a virus, and ChIP-seq experiments reveal genome-wide epigenetic changes in response to infection. Deep mutational scanning is a relatively novel form of protein science which allows the in-depth functional analysis of every nucleotide within a viral gene or genome, revealing regions of importance, flexibility, and mutational potential. In this review, we discuss the application of this technique to RNA viruses including members of the Flaviviridae family, Influenza A Virus and Severe Acute Respiratory Syndrome Coronavirus 2. We also briefly discuss the reverse genetics systems which allow for analysis of viral replication cycles, next-generation sequencing technologies and the bioinformatics tools that facilitate this research.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Mutação/genética , Vírus de RNA/genética , Análise de Sequência de RNA , Biologia Computacional , Biblioteca Gênica , Genoma Viral/genética , Vírus de RNA/classificação , Vírus de RNA/fisiologia , Genética Reversa , Proteínas Virais/genética
11.
BMC Infect Dis ; 21(1): 524, 2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34088268

RESUMO

BACKGROUND: With pandemic of coronavirus disease 2019 (COVID-19), human coronaviruses (HCoVs) have recently attached worldwide attention as essential pathogens in respiratory infection. HCoV-229E has been described as a rare cause of lower respiratory infection in immunocompetent adults. CASE PRESENTATION: We reported a 72-year-old man infected by HCoV-229E with rapid progression to acute respiratory distress syndrome, in conjunction with new onset atrial fibrillation, intensive care unit acquired weakness, and recurrent hospital acquired pneumonia. Clinical and radiological data were continuously collected. The absolute number of peripheral T cells and the level of complement components diminished initially and recovered after 2 months. The patient was successfully treated under intensive support care and discharged from the hospital after 3 months and followed. CONCLUSION: HCoV-229E might an essential causative agent of pulmonary inflammation and extensive lung damage. Supportive treatment was essential to HCoVs infection on account of a long duration of immunological recovery in critical HCoV-229E infection.


Assuntos
Resfriado Comum/diagnóstico , Coronavirus Humano 229E , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/diagnóstico , Idoso , Antibacterianos/uso terapêutico , Líquido da Lavagem Broncoalveolar/virologia , Resfriado Comum/complicações , Resfriado Comum/virologia , Infecções por Coronavirus/complicações , Diabetes Mellitus , Pneumonia Associada a Assistência à Saúde/complicações , Pneumonia Associada a Assistência à Saúde/tratamento farmacológico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pneumonia Viral/tratamento farmacológico
12.
BMC Infect Dis ; 21(1): 531, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34090359

RESUMO

BACKGROUND: Group B Streptococcus (GBS) is an important cause of invasive infection in neonates and infants. Cerebrospinal fluid (CSF) findings and culture may not show evidence of infection early in GBS meningitis. Next-generation sequencing (NGS) has the potential to detect microbial genetic material in patients with infectious diseases. We report two cases of infantile sepsis of GBS meningitis with negative results for CSF culture tests, but positive results for NGS analysis. CASE PRESENTATION: Patient 1 was a 22-day-old male infant diagnosed with sepsis and meningitis. His CSF findings showed pleocytosis, decreased glucose, and increased protein levels. However, CSF and blood culture results at admission were negative. He received a total of 3 weeks of treatment with ampicillin and cefotaxime, and showed clinical improvement. GBS was detected through NGS analysis of CSF collected at admission. Patient 2 was a 51-day-old male infant with sepsis. CSF findings on admission were normal, and blood and CSF cultures were also negative. Intravenous ampicillin and cefotaxime treatment were initiated. Treatment was de-escalated to ampicillin alone because Enterococcus faecalis was cultured from urine. He was discharged after a total of 1 week of antibiotic treatment. Six days after discharge, he was re-hospitalized for sepsis. Blood and CSF cultures were negative, and E. faecalis was again cultured from urine. He received a total of 3 weeks of ampicillin treatment for enterococcal-induced nephritis and did not relapse thereafter. NGS pathogen searches were retrospectively performed on both blood and CSF collected at the first and second admission. GBS was detected in the CSF collected at the first admission, but no significant pathogen was detected in the other samples. Inadequate treatment for GBS meningitis at the first admission may have caused the recurrence of the disease. CONCLUSION: Infantile sepsis may present bacterial meningitis that is not diagnosed by either culture testing or CSF findings. NGS analysis for CSF may be useful for confirming the diagnosis of bacterial meningitis.


Assuntos
Antibacterianos/uso terapêutico , Meningites Bacterianas/diagnóstico , Meningites Bacterianas/tratamento farmacológico , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/tratamento farmacológico , Ampicilina/uso terapêutico , Cefotaxima/uso terapêutico , Líquido Cefalorraquidiano/microbiologia , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Meningites Bacterianas/microbiologia , Estudos Retrospectivos , Sepse/diagnóstico , Sepse/tratamento farmacológico , Sepse/microbiologia , Streptococcus agalactiae/efeitos dos fármacos , Streptococcus agalactiae/genética , Streptococcus agalactiae/isolamento & purificação , Urina/microbiologia
13.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 29(3): 741-750, 2021 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-34105467

RESUMO

OBJECTIVE: To analyze gene expression profile of T cell lymphoma Jurkat cell line treated with paclitaxel by computational biology based on next generation sequencing and to explore the possible molecular mechanism of paclitaxel resistance to T cell lymphoma at gene level. METHODS: IC50 of paclitaxel on Jurkat cell line was determined by CCK-8 assay. Gene expression profile of Jurkat cells treated with paclitaxel was acquired by next generation sequencing technology. Gene microarray data related to human T cell lymphoma were screened from Gene Expression Omnibus (GEO) database (including 720 cases of T cell lymphoma and 153 cases of normal tissues). Combined with the sequencing data, differential expression genes (DEGs) were intersected and screened. DAVID database was used for enrichment analysis of GO function and KEGG pathway to determine and visualize functional entries of DEGs, and protein-protein interactions network of DEGs was drawn. The levels of gene expression were detected and verified by RT-qPCR. RESULTS: CCK-8 results showed that the proliferation of Jurkat cells was inhibited by paclitaxel depended on the concentration apparently. Treated by paclitaxel for 48 h, P<0.05 and |log2(FC)|≥1 were used as filter criteria on the results of RNA Sequencing (RNA-Seq) and GeoChip, 351 DEGs were found from Jurkat cells, including 323 up-regulated genes and 28 down-regulated genes. The GO functional annotation and KEGG pathway enrichment analysis showed that the role of paclitaxel was mainly concentrated in protein heterodimerization activity, nucleosome assembly and transcriptional dysregulation in cancer, etc. The results of RT-qPCR were consistent with those of the sequencing analysis, which verified the reliability of this sequencing. CONCLUSION: Paclitaxel can affect the proliferation and apoptosis of T-cell lymphoma by up-regulating JUN gene, orphan nuclear receptor NR4A family genes and histone family genes.


Assuntos
Linfoma de Células T , Paclitaxel , Biologia Computacional , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reprodutibilidade dos Testes
14.
BMC Genomics ; 22(1): 440, 2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34118871

RESUMO

BACKGROUND: Genomic localized hypermutation regions were found in cancers, which were reported to be related to the prognosis of cancers. This genomic localized hypermutation is quite different from the usual somatic mutations in the frequency of occurrence and genomic density. It is like a mutations "violent storm", which is just what the Greek word "kataegis" means. RESULTS: There are needs for a light-weighted and simple-to-use toolkit to identify and visualize the localized hypermutation regions in genome. Thus we developed the R package "kataegis" to meet these needs. The package used only three steps to identify the genomic hypermutation regions, i.e., i) read in the variation files in standard formats; ii) calculate the inter-mutational distances; iii) identify the hypermutation regions with appropriate parameters, and finally one step to visualize the nucleotide contents and spectra of both the foci and flanking regions, and the genomic landscape of these regions. CONCLUSIONS: The kataegis package is available on Bionconductor/Github ( https://github.com/flosalbizziae/kataegis ), which provides a light-weighted and simple-to-use toolkit for quickly identifying and visualizing the genomic hypermuation regions.


Assuntos
Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Software
15.
Virol J ; 18(1): 110, 2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34078394

RESUMO

BACKGROUND: The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. AIM: Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). METHODS: Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. RESULTS: Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. CONCLUSION: The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable.


Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , SARS-CoV-2/genética , Proteínas do Envelope de Coronavírus/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Limite de Detecção , Poliproteínas/genética , RNA Viral/genética , SARS-CoV-2/isolamento & purificação , Sensibilidade e Especificidade , Proteínas Virais/genética
16.
BMC Infect Dis ; 21(1): 552, 2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112116

RESUMO

BACKGROUND: Children affected by infectious diseases may not always have a detectable infectious etiology. Diagnostic uncertainty can lead to prolonged hospitalizations, inappropriately broad or extended courses of antibiotics, invasive diagnostic procedures, and difficulty predicting the clinical course and outcome. Cell-free plasma next-generation sequencing (cfNGS) can identify viral, bacterial, and fungal infections by detecting pathogen DNA in peripheral blood. This testing modality offers the ability to test for many organisms at once in a shotgun metagenomic approach with a rapid turnaround time. We sought to compare the results of cfNGS to conventional diagnostic test results and describe the impact of cfNGS on clinical care in a diverse pediatric population at a large academic children's hospital. METHODS: We performed a retrospective chart review of hospitalized subjects at a tertiary pediatric hospital to determine the diagnostic yield of cfNGS and its impact on clinical care. RESULTS: We describe the clinical application of results from 142 cfNGS tests in the management of 110 subjects over an 8-month study period. In comparison to conventional testing as a reference standard, cfNGS was found to have a positive percent agreement of 89.6% and negative percent agreement of 52.3%. Furthermore, 32.4% of cfNGS results were directly applied to make a clinical change in management. CONCLUSIONS: We demonstrate the clinically utility of cfNGS in the management of acutely ill children. Future studies, both retrospective and prospective, are needed to clarify the optimal indications for testing.


Assuntos
Doenças Transmissíveis/diagnóstico , Testes Diagnósticos de Rotina/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Hospitais Pediátricos , Adolescente , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Criança , Pré-Escolar , Doenças Transmissíveis/sangue , Testes Diagnósticos de Rotina/normas , Feminino , Humanos , Masculino , Metagenoma , Metagenômica , Estudos Retrospectivos
17.
BMC Bioinformatics ; 22(1): 322, 2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34120589

RESUMO

BACKGROUND: Assessing the nucleosome-forming potential of specific DNA sequences is important for understanding complex chromatin organization. Methods for predicting nucleosome positioning include bioinformatics and biophysical approaches. An advantage of bioinformatics methods, which are based on in vivo nucleosome maps, is the use of natural sequences that may contain previously unknown elements involved in nucleosome positioning in vivo. The accuracy of such prediction attempts reflects the genomic coordinate resolution of the nucleosome maps applied. Nucleosome maps are constructed using micrococcal nuclease digestion followed by high-throughput sequencing (MNase-seq). However, as MNase has a strong preference for A/T-rich sequences, MNase-seq may not be appropriate for this purpose. In addition to MNase-seq-based maps, base pair-resolution chemical maps of in vivo nucleosomes from three different species (budding and fission yeasts, and mice) are currently available. However, these chemical maps have yet to be integrated into publicly available computational methods. RESULTS: We developed a Bioconductor package (named nuCpos) to demonstrate the superiority of chemical maps in predicting nucleosome positioning. The accuracy of chemical map-based prediction in rotational settings was higher than that of the previously developed MNase-seq-based approach. With our method, predicted nucleosome occupancy reasonably matched in vivo observations and was not affected by A/T nucleotide frequency. Effects of genetic alterations on nucleosome positioning that had been observed in living yeast cells could also be predicted. nuCpos calculates individual histone binding affinity (HBA) scores for given 147-bp sequences to examine their suitability for nucleosome formation. We also established local HBA as a new parameter to predict nucleosome formation, which was calculated for 13 overlapping nucleosomal DNA subsequences. HBA and local HBA scores for various sequences agreed well with previous in vitro and in vivo studies. Furthermore, our results suggest that nucleosomal subsegments that are disfavored in different rotational settings contribute to the defined positioning of nucleosomes. CONCLUSIONS: Our results demonstrate that chemical map-based statistical models are beneficial for studying nucleosomal DNA features. Studies employing nuCpos software can enhance understanding of chromatin regulation and the interpretation of genetic alterations and facilitate the design of artificial sequences.


Assuntos
Biologia Computacional , Nuclease do Micrococo , Nucleossomos , Software , Animais , Cromatina , DNA , Sequenciamento de Nucleotídeos em Larga Escala , Camundongos , Nucleossomos/genética
18.
BMC Genomics ; 22(1): 446, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34126923

RESUMO

BACKGROUND: The combination of sodium bisulfite treatment with highly-parallel sequencing is a common method for quantifying DNA methylation across the genome. The power to detect between-group differences in DNA methylation using bisulfite-sequencing approaches is influenced by both experimental (e.g. read depth, missing data and sample size) and biological (e.g. mean level of DNA methylation and difference between groups) parameters. There is, however, no consensus about the optimal thresholds for filtering bisulfite sequencing data with implications for the reproducibility of findings in epigenetic epidemiology. RESULTS: We used a large reduced representation bisulfite sequencing (RRBS) dataset to assess the distribution of read depth across DNA methylation sites and the extent of missing data. To investigate how various study variables influence power to identify DNA methylation differences between groups, we developed a framework for simulating bisulfite sequencing data. As expected, sequencing read depth, group size, and the magnitude of DNA methylation difference between groups all impacted upon statistical power. The influence on power was not dependent on one specific parameter, but reflected the combination of study-specific variables. As a resource to the community, we have developed a tool, POWEREDBiSeq, which utilizes our simulation framework to predict study-specific power for the identification of DNAm differences between groups, taking into account user-defined read depth filtering parameters and the minimum sample size per group. CONCLUSIONS: Our data-driven approach highlights the importance of filtering bisulfite-sequencing data by minimum read depth and illustrates how the choice of threshold is influenced by the specific study design and the expected differences between groups being compared. The POWEREDBiSeq tool, which can be applied to different types of bisulfite sequencing data (e.g. RRBS, whole genome bisulfite sequencing (WGBS), targeted bisulfite sequencing and amplicon-based bisulfite sequencing), can help users identify the level of data filtering needed to optimize power and aims to improve the reproducibility of bisulfite sequencing studies.


Assuntos
Metilação de DNA , Sulfitos , Epigenômica , Sequenciamento de Nucleotídeos em Larga Escala , Reprodutibilidade dos Testes , Análise de Sequência de DNA
19.
BMC Bioinformatics ; 22(1): 323, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34126932

RESUMO

BACKGROUND: Histone modification constitutes a basic mechanism for the genetic regulation of gene expression. In early 2000s, a powerful technique has emerged that couples chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq). This technique provides a direct survey of the DNA regions associated to these modifications. In order to realize the full potential of this technique, increasingly sophisticated statistical algorithms have been developed or adapted to analyze the massive amount of data it generates. Many of these algorithms were built around natural assumptions such as the Poisson distribution to model the noise in the count data. In this work we start from these natural assumptions and show that it is possible to improve upon them. RESULTS: Our comparisons on seven reference datasets of histone modifications (H3K36me3 & H3K4me3) suggest that natural assumptions are not always realistic under application conditions. We show that the unconstrained multiple changepoint detection model with alternative noise assumptions and supervised learning of the penalty parameter reduces the over-dispersion exhibited by count data. These models, implemented in the R package CROCS ( https://github.com/aLiehrmann/CROCS ), detect the peaks more accurately than algorithms which rely on natural assumptions. CONCLUSION: The segmentation models we propose can benefit researchers in the field of epigenetics by providing new high-quality peak prediction tracks for H3K36me3 and H3K4me3 histone modifications.


Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Sequenciamento de Nucleotídeos em Larga Escala , Algoritmos , Imunoprecipitação da Cromatina , Análise de Sequência de DNA
20.
Int J Mol Sci ; 22(9)2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-34064462

RESUMO

MicroRNAs (miRNAs) are regulators of the post-transcription stage of gene activity documented to play central roles in flower and fruit development in model plant species. However, little is known about their roles and differences in domesticated and wild Capsicum species. In this study, we used high-throughput sequencing to analyze the miRNA content at three developmental stages (flower, small fruit, and middle fruit) from two cultivated (C. baccatum and C. annuum) and two wild (C. chacoense and C. eximium) pepper species. This analysis revealed 22 known and 27 novel miRNAs differentially expressed across species and tissues. A number of stage- and species-specific miRNAs were identified, and Gene Ontology terms were assigned to 138 genes targeted by the miRNAs. Most Gene Ontology terms were for the categories "genetic information processing", "signaling and cellular processes", "amino acid metabolism", and "carbohydrate metabolism". Enriched KEGG analysis revealed the pathways amino acids, sugar and nucleotide metabolism, starch and sucrose metabolism, and fructose-mannose metabolism among the principal ones regulated by miRNAs during pepper fruit ripening. We predicted miRNA-target gene interactions regulating flowering time and fruit development, including miR156/157 with SPL genes, miR159 with GaMYB proteins, miR160 with ARF genes, miR172 with AP2-like transcription factors, and miR408 with CLAVATA1 gene across the different Capsicum species. In addition, novel miRNAs play an important role in regulating interactions potentially controlling plant pathogen defense and fruit quality via fructokinase, alpha-L-arabinofuranosidase, and aromatic and neutral amino acid transporter. Overall, the small RNA-sequencing results from this study represent valuable information that provides a solid foundation for uncovering the miRNA-mediated mechanisms of flower and fruit development between domesticated and wild Capsicum species.


Assuntos
Capsicum/genética , Flores/genética , Frutas/genética , Regulação da Expressão Gênica de Plantas , MicroRNAs/genética , Proteínas de Plantas/genética , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Capsicum/classificação , Capsicum/crescimento & desenvolvimento , Capsicum/metabolismo , Domesticação , Flores/crescimento & desenvolvimento , Flores/metabolismo , Frutoquinases/genética , Frutoquinases/metabolismo , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Ontologia Genética , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Redes e Vias Metabólicas/genética , MicroRNAs/classificação , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Proteínas de Plantas/classificação , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Fatores de Transcrição/classificação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...