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1.
Gut ; 69(1): 52-61, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-30971436

RESUMO

OBJECTIVE: Despite improvements in imaging, serum CA19-9 and pathological evaluation, differentiating between benign and malignant bile duct strictures remains a diagnostic conundrum. Recent developments in next-generation sequencing (NGS) have opened new opportunities for early detection and management of cancers but, to date, have not been rigorously applied to biliary specimens. DESIGN: We prospectively evaluated a 28-gene NGS panel (BiliSeq) using endoscopic retrograde cholangiopancreatography-obtained biliary specimens from patients with bile duct strictures. The diagnostic performance of serum CA19-9, pathological evaluation and BiliSeq was assessed on 252 patients (57 trainings and 195 validations) with 346 biliary specimens. RESULTS: The sensitivity and specificity of BiliSeq for malignant strictures was 73% and 100%, respectively. In comparison, an elevated serum CA19-9 and pathological evaluation had sensitivities of 76% and 48%, and specificities of 69% and 99%, respectively. The combination of BiliSeq and pathological evaluation increased the sensitivity to 83% and maintained a specificity of 99%. BiliSeq improved the sensitivity of pathological evaluation for malignancy from 35% to 77% for biliary brushings and from 52% to 83% for biliary biopsies. Among patients with primary sclerosing cholangitis (PSC), BiliSeq had an 83% sensitivity as compared with pathological evaluation with an 8% sensitivity. Therapeutically relevant genomic alterations were identified in 20 (8%) patients. Two patients with ERBB2-amplified cholangiocarcinoma received a trastuzumab-based regimen and had measurable clinicoradiographic response. CONCLUSIONS: The combination of BiliSeq and pathological evaluation of biliary specimens increased the detection of malignant strictures, particularly in patients with PSC. Additionally, BiliSeq identified alterations that may stratify patients for specific anticancer therapies.


Assuntos
Colangiopancreatografia Retrógrada Endoscópica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/diagnóstico , Neoplasias dos Ductos Biliares/genética , Neoplasias dos Ductos Biliares/patologia , Doenças Biliares/diagnóstico , Doenças Biliares/genética , Doenças Biliares/patologia , Biomarcadores Tumorais/sangue , Antígeno CA-19-9/sangue , Constrição Patológica/diagnóstico , Constrição Patológica/genética , Diagnóstico Diferencial , Feminino , Humanos , Cirrose Hepática Biliar/diagnóstico , Cirrose Hepática Biliar/genética , Cirrose Hepática Biliar/patologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Adulto Jovem
2.
Gene ; 725: 144170, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31647996

RESUMO

Caragana korshinskii Kom. is a legume shrub that is widely distributed across desert habitats with gravely, sandy, and saline soils in Asia and Africa. C. korshinskii has highly developed roots and a strong tolerance to abiotic stress. At present, there are few genetic studies of C. korshinskii because of the limited availability of genomic resources. To understand the comprehensive mechanisms that are associated with drought tolerance, we used RNA-seq to survey the differentially expressed genes (DEGs) in comparisons of drought-treated and control plants. After analysing the sequencing results, we found 440 differentially expressed genes existing in drought-treated and control plants. Among the DEGs, 39 unigenes showed up-regulated expression after drought treatment, while 401 unigenes were down-regulated. We used the KEGG database to annotate these drought-induced genes; 126 unigenes were identified by KEGG pathway annotation, and approximately 28% of the unigenes with known function fell into categories related to fatty acid metabolism, starch, sucrose metabolism, and nitrogen metabolism, suggesting that these pathways or processes may be involved in the drought response. Finally, we confirmed that one gene has a potential function in drought tolerance. Our study is the first to provide transcriptomic resources for Caragana korshinskii and to determine its digital gene expression profile under conditions of drought stress using the assembled transcriptomic data for reference. These data provide a valuable resource for genetic and genomic studies of desert plants under abiotic stress conditions.


Assuntos
Caragana/genética , Perfilação da Expressão Gênica/métodos , Estresse Fisiológico/genética , Secas , Fabaceae/genética , Regulação da Expressão Gênica de Plantas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular , RNA/genética , Transcriptoma/genética
3.
Zhonghua Fu Chan Ke Za Zhi ; 54(12): 808-814, 2019 Dec 25.
Artigo em Chinês | MEDLINE | ID: mdl-31874470

RESUMO

Objective: To evaluate the application of combinatorial probe anchor synthesis (cPAS)-based high-throughput low coverage whole genome sequencing in chromosomal aberration detection in spontaneous miscarriage. Methods: From September 2015 to May 2017, spontaneous miscarriage samples were collected from Inner Mongolia Maternal and Child Health Care Hospital. Those samples were further analyzed with two independent methods, fluorescence in situ hybridization (FISH) and low coverage whole genome sequencing on the BGISEQ-500 high-throughput platform. The performance of low coverage whole genome sequencing was assessed by comparing to FISH results. Results: In 595 spontaneous miscarried specimens, low coverage whole genome sequencing revealed 144 cases (24.2%, 144/595) chromosomal abnormalities, of which a subset of 137 cases (23.0%, 137/595) were detected as aneuploidies, 2 cases (0.3%, 2/595) as mosaicisms and 5 cases (0.8%, 5/595) as copy number variation (≥5 Mb). Conclusion: cPAS-based high-throughput low coverage whole genome sequencing is a reliable method in detecting chromosomal aberrations inspontaneous abortion tissues, including chromosome aneuploidies, mosaicisms and copy number variation (≥5 Mb).


Assuntos
Aborto Espontâneo/genética , Aberrações Cromossômicas/estatística & dados numéricos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Criança , China , Aberrações Cromossômicas/embriologia , Cromossomos/genética , DNA/genética , Variações do Número de Cópias de DNA/genética , Feminino , Humanos , Hibridização in Situ Fluorescente , Gravidez , Sequenciamento Completo do Genoma/métodos
4.
BMC Bioinformatics ; 20(Suppl 24): 596, 2019 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-31861975

RESUMO

BACKGROUND: Adenosine-to-inosine RNA editing can markedly diversify the transcriptome, leading to a variety of critical molecular and biological processes in mammals. Over the past several years, researchers have developed several new pipelines and software packages to identify RNA editing sites with a focus on downstream statistical analysis and functional interpretation. RESULTS: Here, we developed a user-friendly public webserver named MIRIA that integrates statistics and visualization techniques to facilitate the comprehensive analysis of RNA editing sites data identified by the pipelines and software packages. MIRIA is unique in that provides several analytical functions, including RNA editing type statistics, genomic feature annotations, editing level statistics, genome-wide distribution of RNA editing sites, tissue-specific analysis and conservation analysis. We collected high-throughput RNA sequencing (RNA-seq) data from eight tissues across seven species as the experimental data for MIRIA and constructed an example result page. CONCLUSION: MIRIA provides both visualization and analysis of mammal RNA editing data for experimental biologists who are interested in revealing the functions of RNA editing sites. MIRIA is freely available at https://mammal.deepomics.org.


Assuntos
Mamíferos , Edição de RNA , Análise de Sequência de RNA , Transcriptoma , Animais , Genoma , Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mamíferos/genética , RNA/genética , Análise de Sequência de RNA/métodos
5.
Medicine (Baltimore) ; 98(42): e17601, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31626137

RESUMO

BACKGROUND: Lung adenocarcinoma (LA) is a most common form of non-small cell lung cancer (NSCLC). To date, there are still no effective early diagnosis methods for patients to be cured in time. Noncoding RNA plays an important role in oncogenesis and tumor development. The expression profile of circular RNA (circRNA) in peripheral whole blood (PWB) of LA has not been systematically investigated. In this study, we identified the differentially expressed (DE) circRNAs in PWB of LA by high-throughput sequencing. METHODS: Five paired LA and normal participants PWB samples were chosen to investigate the expression profile of circRNAs by high-throughput sequencing. Twenty LA and 10 normal controls PWB samples were subjected to reverse-transcription polymerase chain reaction (RT-PCR) for validation of circRNAs expression profile. Gene Ontology (GO) functional analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and circRNA-miRNA network analysis was also performed to predict the function of circRNAs in PWB. RESULTS: A total of 10566 circRNAs were identified and annotated, most of the circRNAs were exonic (78.14%). Statistical analysis revealed 4390 DE circRNAs, in which were 3009 upregulated circRNAs and1381downregulated circRNAs in LA. RT-PCR results showed that circRNA expression in LA was higher than that in controls. GO functional analysis, KEGG pathway analysis, and circRNA-miRNA network analysis all showed that circRNAs correlated with tumor development and progression to a certain degree. The current study is the first to systematically characterize and annotate circRNA expression in PWB of LA. Some host genes of the DE circRNAs were involved in tumor signaling pathway and had complicated correlations with tumor related miRNAs, indicating that circRNAs might involve in development and progression of LA. CONCLUSIONS: Our study revealed that circRNAs were abnormally expressed in PWB of LA, which might offer potential targets for the early diagnosis of the disease and new genetic insights into LA.


Assuntos
Adenocarcinoma de Pulmão/genética , Regulação Neoplásica da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Adenocarcinoma de Pulmão/sangue , Perfilação da Expressão Gênica/métodos , Humanos , RNA/biossíntese , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para Cima
6.
Nat Biotechnol ; 37(10): 1229-1236, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31501560

RESUMO

The density and long-term stability of DNA make it an appealing storage medium, particularly for long-term data archiving. Existing DNA storage technologies involve the synthesis and sequencing of multiple nominally identical molecules in parallel, resulting in information redundancy. We report the development of encoding and decoding methods that exploit this redundancy using composite DNA letters. A composite DNA letter is a representation of a position in a sequence that consists of a mixture of all four DNA nucleotides in a predetermined ratio. Our methods encode data using fewer synthesis cycles. We encode 6.4 MB into composite DNA, with distinguishable composition medians, using 20% fewer synthesis cycles per unit of data, as compared to previous reports. We also simulate encoding with larger composite alphabets, with distinguishable composition deciles, to show that 75% fewer synthesis cycles are potentially sufficient. We describe applicable error-correcting codes and inference methods, and investigate error patterns in the context of composite DNA letters.


Assuntos
DNA/síntese química , Algoritmos , Sequência de Bases , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Armazenamento e Recuperação da Informação , Análise de Sequência de DNA/métodos
7.
Arch Virol ; 164(12): 3103-3106, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31520218

RESUMO

A large contig with sequence similarities to members of the genus Robigovirus was identified by high-throughput sequencing analysis from a symptomless cherry accession. The complete genome sequence of this new virus is 8,384 nucleotides in length, excluding the 3' poly(A) tail. Its genome organization is very similar to those of four known robigoviruses, encoding a putative replicase, three 'triple gene block' proteins, a coat protein, and an unknown protein, 2a. Unlike the four cherry robigoviruses, the new virus does not contain a putative ORF5a. The full-length genome of the virus, which is provisionally named "cherry robigovirus 5" (CRV-5), is 52-57% identical to genome sequences of other robigoviruses. Phylogenetic analysis showed that CRV-5 and other robigoviruses group in a cluster, supporting its assignment to a new species in the genus Robigovirus.


Assuntos
Flexiviridae/classificação , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Prunus/virologia , Flexiviridae/genética , Flexiviridae/isolamento & purificação , Tamanho do Genoma , Fases de Leitura Aberta , Filogenia , Análise de Sequência de RNA , Homologia de Sequência do Ácido Nucleico
8.
Nat Protoc ; 14(10): 2749-2780, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31471598

RESUMO

Circulating cell-free DNA (cfDNA) comprises small DNA fragments derived from normal and tumor tissue that are released into the bloodstream. Recently, methylation profiling of cfDNA as a liquid biopsy tool has been gaining prominence due to the presence of tissue-specific markers in cfDNA. We have previously reported cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) as a sensitive, low-input, cost-efficient and bisulfite-free approach to profiling DNA methylomes of plasma cfDNA. cfMeDIP-seq is an extension of a previously published MeDIP-seq protocol and is adapted to allow for methylome profiling of samples with low input (ranging from 1 to 10 ng) of DNA, which is enabled by the addition of 'filler DNA' before immunoprecipitation. This protocol is not limited to plasma cfDNA; it can also be applied to other samples that are naturally sheared and at low availability (e.g., urinary cfDNA and cerebrospinal fluid cfDNA), and is potentially applicable to other applications beyond cancer detection, including prenatal diagnostics, cardiology and monitoring of immune response. The protocol presented here should enable any standard molecular laboratory to generate cfMeDIP-seq libraries from plasma cfDNA in ~3-4 d.


Assuntos
Ácidos Nucleicos Livres/análise , Ácidos Nucleicos Livres/metabolismo , Metilação de DNA , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Ácidos Nucleicos Livres/isolamento & purificação , Biologia Computacional/métodos , Biblioteca Gênica , Humanos , Imunoprecipitação , Fluxo de Trabalho
9.
Yonsei Med J ; 60(10): 914-923, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31538426

RESUMO

PURPOSE: Few efforts have been made to integrate a next generation sequencing (NGS) panel into standard clinical treatment of ovarian cancer. The aim of this study was to investigate the clinical utility of NGS and to identify clinically impactful information beyond targetable alterations. MATERIALS AND METHODS: We conducted a retrospective review of 84 patients with ovarian cancer who underwent NGS between March 1, 2017, and July 31, 2018, at the Yonsei Cancer Hospital. We extracted DNA from formalin-fixed, paraffin-embedded tissue samples of ovarian cancer. The TruSight Tumor 170 gene panel was used to prepare libraries, and the MiSeq instrument was used for NGS. RESULTS: Of the 84 patients, 55 (65.1%) had high-grade serous carcinomas. Seventy-three (86.7%) patients underwent NGS at the time of diagnosis, and 11 (13.3%) underwent NGS upon relapse. The most common genetic alterations were in TP53 (64%), PIK3CA (15%), and BRCA1/2 (13%), arising as single nucleotide variants and indels. MYC amplification (27%) was the most common copy number variation and fusion. Fifty-seven (67.9%) patients had more than one actionable alteration other than TP53. Seven (8.3%) cases received matched-target therapy based on the following sequencing results: BRCA1 or 2 mutation, poly ADP ribose polymerase inhibitor (n=5); PIK3CA mutation, AKT inhibitor (n=1); and MLH1 mutation, PD-1 inhibitor (n=1). Fifty-three (63.0%) patients had a possibility of treatment change, and 8 (9.5%) patients received genetic counseling. CONCLUSION: Implementation of NGS may help in identifying patients who might benefit from targeted treatment therapies and genetic counseling.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Neoplasias Ovarianas/genética , Padrões de Prática Médica , Adulto , Idoso , Variações do Número de Cópias de DNA/genética , Feminino , Genoma Humano , Humanos , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Mutação/genética , Neoplasias Ovarianas/patologia , Estudos Retrospectivos
10.
Chem Commun (Camb) ; 55(70): 10404-10407, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31402361

RESUMO

We established an efficient method for single-cell miRNA analysis by droplet microfluidics, which has high sensitivity of single molecule detection and high throughput. Single-cell analysis of multiple miRNAs in various cells shows that miRNA expression is closely related to cancer type. CTC analysis shows that the method is applicable for rare cell analysis.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , MicroRNAs/genética , Neoplasias/genética , Análise de Célula Única , Linhagem Celular Tumoral , Fluorescência , Humanos , Limite de Detecção
11.
Cancer Sci ; 110(10): 3038-3048, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31385405

RESUMO

Retroperitoneal liposarcoma (RLPS) is one of the most common subtypes of retroperitoneal soft tissue sarcomas and lacks effective treatment. This study aimed to provide a thorough profile of immune characteristics of RLPS. This study included 56 RLPS patients. Multisite tumor tissues were collected from 16 patients. Immunohistochemistry was carried out to identify CD4+ , CD8+ , FoxP3+ , CD20+ , or programmed cell death-1 (PD-1)+ tumor infiltrating lymphocytes (TILs) and  Programmed cell death ligand-1 (PD-L1) expression in tumor tissues. Ultradeep sequencing of T-cell receptor (TCR) ß-chain gene was carried out in 42 tumor samples as well as peripheral blood samples collected from 6 patients. In RLPS, TILs were distributed in 3 patterns and T cells were more prevalent than B cells. Generally, the proportion of TILs decreased and PD-L1 expression increased with tumor progression. Patients with higher PD-1/PD-L1 expression tended to have poorer prognosis, whereas patients with tertiary lymphoid structure tended to have a favorable disease-free survival. Although T-cell clones in tumors were quite different from those in peripheral blood, TCR sequencing showed low TCR repertoire reads as well as polyclonal status within tumors, which indicated limited T cell response in the tumors. Both TILs distribution and TCR repertoires suggested spatial immune heterogeneity in RLPS. Our research described the immune landscape of RLPS, and suggested RLPS might be a kind of tumor with low T cell infiltration as well as great immune heterogeneity. Therefore, strategies that can facilitate lymphocytic infiltration and immune reactivity need to be developed in the future to improve the efficacy of immunotherapy.


Assuntos
Antígeno B7-H1/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Lipossarcoma/imunologia , Linfócitos do Interstício Tumoral/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Neoplasias Retroperitoneais/imunologia , Adulto , Idoso , Linfócitos B/imunologia , Intervalo Livre de Doença , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Lipossarcoma/genética , Lipossarcoma/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Neoplasias Retroperitoneais/genética , Neoplasias Retroperitoneais/mortalidade , Análise de Sequência de DNA , Linfócitos T/imunologia , Regulação para Cima
12.
Genome Biol ; 20(1): 155, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387612

RESUMO

We describe a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts or genomic regions from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, which are all pre-selected and optimized in silico. By applying the matrices in a novel workflow named Barcode Assembly foR Targeted Sequencing (BART-Seq), we analyze developmental states of thousands of single human pluripotent stem cells, either in different maintenance media or upon Wnt/ß-catenin pathway activation, which identifies the mechanisms of differentiation induction. Moreover, we apply BART-Seq to the genetic screening of breast cancer patients and identify BRCA mutations with very high precision. The processing of thousands of samples and dynamic range measurements that outperform global transcriptomics techniques makes BART-Seq first targeted sequencing technique suitable for numerous research applications.


Assuntos
Perfilação da Expressão Gênica/métodos , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Neoplasias da Mama/genética , Análise Custo-Benefício , Células-Tronco Embrionárias/metabolismo , Feminino , Perfilação da Expressão Gênica/economia , Genômica/economia , Sequenciamento de Nucleotídeos em Larga Escala/economia , Humanos , Células-Tronco Pluripotentes/metabolismo , Análise de Sequência de RNA/economia , Análise de Célula Única/economia , Análise de Célula Única/métodos , Via de Sinalização Wnt , Fluxo de Trabalho
13.
Nat Biotechnol ; 37(10): 1155-1162, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31406327

RESUMO

The DNA sequencing technologies in use today produce either highly accurate short reads or less-accurate long reads. We report the optimization of circular consensus sequencing (CCS) to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb). We applied our approach to sequence the well-characterized human HG002/NA24385 genome and obtained precision and recall rates of at least 99.91% for single-nucleotide variants (SNVs), 95.98% for insertions and deletions <50 bp (indels) and 95.99% for structural variants. Our CCS method matches or exceeds the ability of short-read sequencing to detect small variants and structural variants. We estimate that 2,434 discordances are correctable mistakes in the 'genome in a bottle' (GIAB) benchmark set. Nearly all (99.64%) variants can be phased into haplotypes, further improving variant detection. De novo genome assembly using CCS reads alone produced a contiguous and accurate genome with a contig N50 of >15 megabases (Mb) and concordance of 99.997%, substantially outperforming assembly with less-accurate long reads.


Assuntos
DNA Circular/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Sequência de Bases , Variação Genética , Haplótipos , Humanos
14.
Nucleic Acids Res ; 47(18): e105, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31372651

RESUMO

A primary challenge in the analysis of RNA-seq data is to identify differentially expressed genes or transcripts while controlling for technical biases. Ideally, a statistical testing procedure should incorporate the inherent uncertainty of the abundance estimates arising from the quantification step. Most popular methods for RNA-seq differential expression analysis fit a parametric model to the counts for each gene or transcript, and a subset of methods can incorporate uncertainty. Previous work has shown that nonparametric models for RNA-seq differential expression may have better control of the false discovery rate, and adapt well to new data types without requiring reformulation of a parametric model. Existing nonparametric models do not take into account inferential uncertainty, leading to an inflated false discovery rate, in particular at the transcript level. We propose a nonparametric model for differential expression analysis using inferential replicate counts, extending the existing SAMseq method to account for inferential uncertainty. We compare our method, Swish, with popular differential expression analysis methods. Swish has improved control of the false discovery rate, in particular for transcripts with high inferential uncertainty. We apply Swish to a single-cell RNA-seq dataset, assessing differential expression between sub-populations of cells, and compare its performance to the Wilcoxon test.


Assuntos
Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Algoritmos , Linhagem da Célula/genética , Expressão Gênica/genética , Humanos , RNA/genética , Software
15.
Nucleic Acids Res ; 47(18): e111, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31372654

RESUMO

A key challenge in modeling single-cell RNA-seq data is to capture the diversity of gene expression states regulated by different transcriptional regulatory inputs across individual cells, which is further complicated by largely observed zero and low expressions. We developed a left truncated mixture Gaussian (LTMG) model, from the kinetic relationships of the transcriptional regulatory inputs, mRNA metabolism and abundance in single cells. LTMG infers the expression multi-modalities across single cells, meanwhile, the dropouts and low expressions are treated as left truncated. We demonstrated that LTMG has significantly better goodness of fitting on an extensive number of scRNA-seq data, comparing to three other state-of-the-art models. Our biological assumption of the low non-zero expressions, rationality of the multimodality setting, and the capability of LTMG in extracting expression states specific to cell types or functions, are validated on independent experimental data sets. A differential gene expression test and a co-regulation module identification method are further developed. We experimentally validated that our differential expression test has higher sensitivity and specificity, compared with other five popular methods. The co-regulation analysis is capable of retrieving gene co-regulation modules corresponding to perturbed transcriptional regulations. A user-friendly R package with all the analysis power is available at https://github.com/zy26/LTMGSCA.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA/genética , Análise de Célula Única/métodos , Software , Algoritmos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Modelos Estatísticos , Análise de Sequência de RNA/métodos
16.
Medicine (Baltimore) ; 98(35): e16985, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31464947

RESUMO

RATIONALE: Angiostrongylus cantonensis-induced eosinophilic meningoencephalitis (AEM) in infants is a very rare but fatal disease. Utilization of genetic assay to detect the cerebral parasite plays an important role for the treatment of the infection. PATIENT CONCERNS: Two infants (<2 years) presented with cough, intermittent fever, mental fatigue, and poor diet. DIAGNOSIS: The patients were under clinical examination and laboratory test including cardiac ultrasound, chest X-ray, blood or cerebrospinal fluid (CSF) cell counting, serum enzyme-linked immunosorbent assay (ELISA), head magnetic resonance imaging (MRI) and next-generation sequencing (NGS) on DNA from CSF. Due to hypereosinophils in patients' peripheral blood and CSF, and abundant DNA sequences from A cantonensis in CSF, the patients were diagnosed with Angiostrongylus eosinophilic meningoencephalitis. INTERVENTIONS: The patients were treated with albendazole to deworm, and methylprednisolone to reduce inflammation. OUTCOME: The patients were completely recovered from AEM without relapse after 10-day treatment. LESSONS: ELISA and MRI are not sufficiently accurate for the diagnosis of AEM in infants. NGS can specify the infection by the cerebral parasite and offers a new effective approach for the early and precise diagnosis of AEM in infants.


Assuntos
Eosinofilia/complicações , Meningoencefalite/complicações , Meningoencefalite/diagnóstico , Meningoencefalite/parasitologia , Infecções por Strongylida/diagnóstico , Albendazol/uso terapêutico , Angiostrongylus cantonensis , Animais , Anti-Helmínticos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Ensaio de Imunoadsorção Enzimática , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lactente , Imagem por Ressonância Magnética , Masculino , Meningoencefalite/tratamento farmacológico , Metilprednisolona/uso terapêutico , Infecções por Strongylida/tratamento farmacológico , Infecções por Strongylida/parasitologia
17.
Presse Med ; 48(7-8 Pt 1): 816-824, 2019.
Artigo em Francês | MEDLINE | ID: mdl-31439443

RESUMO

Diagnosis of mature B cell malignancies is highly multidisciplinary. Biological tools provide diagnostic, prognostic and theranostic information. Biological hematology allows considering mature B cell diseases from two perspectives : cellular and molecular approaches. Cytomorphology and flow cytometry are tools from cell hematology. Conventional cytogenetics, FISH and molecular biology are tools from molecular hematology. NGS is a new technique that could dramatically change diagnostic and therapeutic management of B cell malignancies in the near future. Integration of clinical, pathological and biological data allows for personalized management of these diseases.


Assuntos
Técnicas de Laboratório Clínico/métodos , Leucemia de Células B/diagnóstico , Linfoma de Células B/diagnóstico , Integração de Sistemas , Hibridização Genômica Comparativa/métodos , Citodiagnóstico/métodos , Análise Citogenética/métodos , Análise Mutacional de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Imunofenotipagem/métodos , Hibridização in Situ Fluorescente/métodos , Leucemia de Células B/genética , Leucemia de Células B/patologia , Linfoma de Células B/genética , Linfoma de Células B/patologia , Técnicas de Diagnóstico Molecular/métodos , Imagem Multimodal/métodos , Imagem Multimodal/tendências
18.
BMC Bioinformatics ; 20(1): 424, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31416440

RESUMO

BACKGROUND: High throughput DNA/RNA sequencing has revolutionized biological and clinical research. Sequencing is widely used, and generates very large amounts of data, mainly due to reduced cost and advanced technologies. Quickly assessing the quality of giga-to-tera base levels of sequencing data has become a routine but important task. Identification and elimination of low-quality sequence data is crucial for reliability of downstream analysis results. There is a need for a high-speed tool that uses optimized parallel programming for batch processing and simply gauges the quality of sequencing data from multiple datasets independent of any other processing steps. RESULTS: FQStat is a stand-alone, platform-independent software tool that assesses the quality of FASTQ files using parallel programming. Based on the machine architecture and input data, FQStat automatically determines the number of cores and the amount of memory to be allocated per file for optimum performance. Our results indicate that in a core-limited case, core assignment overhead exceeds the benefit of additional cores. In a core-unlimited case, there is a saturation point reached in performance by increasingly assigning additional cores per file. We also show that memory allocation per file has a lower priority in performance when compared to the allocation of cores. FQStat's output is summarized in HTML web page, tab-delimited text file, and high-resolution image formats. FQStat calculates and plots read count, read length, quality score, and high-quality base statistics. FQStat identifies and marks low-quality sequencing data to suggest removal from downstream analysis. We applied FQStat on real sequencing data to optimize performance and to demonstrate its capabilities. We also compared FQStat's performance to similar quality control (QC) tools that utilize parallel programming and attained improvements in run time. CONCLUSIONS: FQStat is a user-friendly tool with a graphical interface that employs a parallel programming architecture and automatically optimizes its performance to generate quality control statistics for sequencing data. Unlike existing tools, these statistics are calculated for multiple datasets and separately at the "lane," "sample," and "experiment" level to identify subsets of the samples with low quality, thereby preventing the loss of complete samples when reliable data can still be obtained.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Software , Bases de Dados de Ácidos Nucleicos , Humanos , Controle de Qualidade , Análise de Sequência de DNA , Análise de Sequência de RNA , Fatores de Tempo
19.
Gene ; 719: 144075, 2019 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-31449843

RESUMO

The human L-DOPA decarboxylase (DDC) is a gene that has been in the center of research attention in many laboratories the last decades, due to its major implication in various disorders, including many types of cancer. In the current work, we used in-house developed RACE and high-throughput sequencing approaches, in order to detect and identify novel DDC transcripts. Bioinformatic analysis revealed new alternative splicing events that support the existence of novel DDC transcripts. As a result, a total of 14 DDC splice variants were identified and their expression profile was investigated in a wide panel of human cancer cell lines. From all 14 novel DDC transcripts that were identified, 9 transcripts are predicted to encode new protein isoforms, while the remaining 5 are nonsense-mediated mRNA decay (NMD) candidates. Our results demonstrate that the human DDC gene undergoes complex processing leading to the figuration of multiple mRNA isoforms in cancer cells.


Assuntos
Processamento Alternativo , Descarboxilases de Aminoácido-L-Aromático/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequência de Bases , Linhagem Celular Tumoral , Éxons , Perfilação da Expressão Gênica , Humanos , RNA Mensageiro/genética
20.
Gene ; 720: 144056, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31437466

RESUMO

Repeated implantation failure (RIF) was mainly due to poor endometrium receptivity. Long noncoding RNAs (lncRNAs) could regulate endometrium receptivity and act in competing endogenous RNA (ceRNA) theory. However, the regulatory mechanism of the lncRNA-miRNA-mRNA network in repeated implantation failure (RIF) is unclear. We obtained RIF-related expression profiles of lncRNAs, mRNAs, and miRNAs using mid-secretory endometrial tissue samples from 5 women with RIF and 5 controls by RNA-sequencing. Co-expression analysis revealed that three functional modules were enriched in immune response/inflammation process; two functional modules were enriched in metabolic/ biosynthetic process, and one functional module were enriched in cell cycle pathway. By adding the miRNA data, ceRNA regulatory relationship of each module was reconstructed. The ceRNA network of the whole differentially expressed RNAs revealed 10 hub lncRNAs. Among them, TRG-AS1, SIMM25, and NEAT1 were involved in the module1, module2, and module3, respectively; LNC00511 and SLC26A4-AS1 in the module4; H19 in the module5. The real-time polymerase chain reaction (RT-PCR) results of 15 randomly selected RNAs were consistent with our sequencing data. These can be used as novel potential biomarkers for RIF. Furthermore, they might be involved in endometrium receptivity by acting as ceRNA.


Assuntos
Biomarcadores/metabolismo , Endométrio/metabolismo , Redes Reguladoras de Genes , Genoma Humano , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Adulto , Estudos de Casos e Controles , Implantação do Embrião , Endométrio/patologia , Feminino , Fertilização In Vitro , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos
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