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1.
Nat Commun ; 12(1): 1780, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741979

RESUMEN

Advanced non-small-cell lung cancer (NSCLC) patients with EGFR T790M-positive tumours benefit from osimertinib, an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). Here we show that the size of the EGFR T790M-positive clone impacts response to osimertinib. T790M subclonality, as assessed by a retrospective NGS analysis of 289 baseline plasma ctDNA samples from T790M-positive advanced NSCLC patients from the AURA3 phase III trial, is associated with shorter progression-free survival (PFS), both in the osimertinib and the chemotherapy-treated patients. Both baseline and longitudinal ctDNA profiling indicate that the T790M subclonal tumours are enriched for PIK3CA alterations, which we demonstrate to confer resistance to osimertinib in vitro that can be partially reversed by PI3K pathway inhibitors. Overall, our results elucidate the impact of tumour heterogeneity on response to osimertinib in advanced stage NSCLC patients and could help define appropriate combination therapies in these patients.


Asunto(s)
Acrilamidas/uso terapéutico , Compuestos de Anilina/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Mutación Missense , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , ADN Tumoral Circulante/análisis , ADN Tumoral Circulante/genética , Fosfatidilinositol 3-Quinasa Clase I/genética , Receptores ErbB/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Inhibidores de Proteínas Quinasas/uso terapéutico , Estudios Retrospectivos
2.
Front Cell Infect Microbiol ; 11: 632490, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33777844

RESUMEN

The novel coronavirus SARS-CoV-2 (causing the disease COVID-19) has caused a highly transmissible and ongoing pandemic worldwide. Due to its rapid development, next-generation sequencing plays vital roles in many aspects. Here, we summarize the current knowledge on the origin and human transmission of SARS-CoV-2 based on NGS analysis. The ACE2 expression levels in various human tissues and relevant cells were compared to provide insights into the mechanism of SAS-CoV-2 infection. Gut microbiota dysbiosis observed by metagenome sequencing and the immunogenetics of COVID-19 patients according to single-cell sequencing analysis were also highlighted. Overall, the application of these sequencing techniques could be meaningful for finding novel intermediate SARS-CoV-2 hosts to block interspecies transmission. This information will further benefit SARS-CoV-2 diagnostic development and new therapeutic target discovery. The extensive application of NGS will provide powerful support for our fight against future public health emergencies.


Asunto(s)
/epidemiología , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , /genética , /genética , /microbiología , /virología , Disbiosis/virología , Microbioma Gastrointestinal , Humanos , Metagenoma , Pandemias
3.
Anticancer Res ; 41(3): 1341-1348, 2021 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-33788725

RESUMEN

BACKGROUND/AIM: Cancer profiling tests using formalin-fixed paraffin-embedded (FFPE) specimens with various conditions have become an essential tool for cancer treatment. The robustness of these tests needs to be addressed. MATERIALS AND METHODS: A cancer profiling test, NCC oncopanel, was tested with FFPE specimens from various tissues with different storage conditions and fixation lengths. Next generation sequencing was performed with Miseq and the data were assembled using the human reference genome hg19. RESULTS: Duration of storage and fixation affected the mapping statistics. Prolonged storage increased outward read paring and longer fixation rates caused increased singletons and unmapped reads. CONCLUSION: Our results indicate that a cancer profiling test with target capturing method, NCC oncopanel, shows robustness for FFPE cancer specimens with various storage conditions.


Asunto(s)
Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/genética , Adhesión en Parafina/métodos , Manejo de Especímenes/métodos , Fijación del Tejido/métodos , Fijadores/química , Formaldehído/química , Genómica/métodos , Humanos , Mutación , Neoplasias/patología
4.
Methods Mol Biol ; 2278: 21-29, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33649945

RESUMEN

Rapid and efficient protocols aimed at the isolation and purification of DNA for the purpose of downstream applications, such as cloning, PCR, Southern blotting, or sequencing, are essential for genetic, biochemical, and molecular biological analyses of a given bacterium. The protocols herein presented provide a robust and efficient method for the isolation of chromosomal and plasmid DNA from Bifidobacterium strains by organic extraction. The methods are simple, and the yield, purity, and quality of the DNA are adequate to perform various downstream applications including next-generation sequencing.


Asunto(s)
Bifidobacterium/genética , ADN Bacteriano/genética , Plásmidos/genética , Cromosomas Bacterianos/genética , ADN Bacteriano/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Plásmidos/aislamiento & purificación , Secuenciación Completa del Genoma/métodos
5.
Methods Mol Biol ; 2278: 31-44, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33649946

RESUMEN

Genome assembly and annotation are two of the key actions that must be undertaken in order to explore the genomic repertoire of (bifido)bacteria. The gathered information can be employed to genomically characterize a given microorganism, and can also be used to perform comparative genome analysis by including other sequenced (bifido)bacterial strains. Here, we highlight various bioinformatic programs able to manage next generation sequencing data starting from the assembly of a genome to the comparative analyses between strains.


Asunto(s)
Bifidobacterium/genética , Genoma Bacteriano , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Anotación de Secuencia Molecular/métodos , Filogenia , Análisis de Secuencia de ADN/métodos , Secuenciación Completa del Genoma/métodos
6.
Methods Mol Biol ; 2278: 71-85, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33649949

RESUMEN

Bifidobacteria are important early colonizers of the human intestinal tract. The relative abundance of bifidobacterial species may be modulated, in part, by bacteriophage activity. Metagenomic studies of these populations is a crucial step in understanding this important interaction. This chapter outlines the technical instructions required to analyze the virome of a bifidobacteria-rich sample, for example, an infant fecal sample.


Asunto(s)
Bacteriófagos/genética , Bifidobacterium/virología , Heces/microbiología , ADN Viral/genética , Microbioma Gastrointestinal , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Lactante
7.
Methods Mol Biol ; 2292: 95-104, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33651354

RESUMEN

Application of next generation sequencing techniques in the field of liquid biopsy, in particular urine, requires specific bioinformatics methods in order to deal with its peculiarity. Many aspects of cancer can be explored starting from nucleic acids, especially from cell-free DNA and circulating tumor DNA in order to characterize cancer. It is possible to detect small mutations, as single nucleotide variants, small insertions and deletions, copy-number alterations, and epigenetic profiles. Due to the low fraction of circulating tumor DNA over the whole cell-free DNA, some methods have been exploited. One of them is the application of unique barcodes to each DNA fragment in order to lower the limit of detection of cancer-related variants. Some bioinformatics workflows and tools are the same of a classic analysis of tumor tissue, but there are some steps in which specific algorithms have to be introduced.


Asunto(s)
Ácidos Nucleicos Libres de Células/orina , Neoplasias/orina , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/orina , Ácidos Nucleicos Libres de Células/genética , Variaciones en el Número de Copia de ADN , Metilación de ADN , Epigénesis Genética , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Biopsia Líquida/métodos , Neoplasias/genética
8.
Nat Commun ; 12(1): 1405, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658502

RESUMEN

Population scale sweeps of viral pathogens, such as SARS-CoV-2, require high intensity testing for effective management. Here, we describe "Systematic Parallel Analysis of RNA coupled to Sequencing for Covid-19 screening" (C19-SPAR-Seq), a multiplexed, scalable, readily automated platform for SARS-CoV-2 detection that is capable of analyzing tens of thousands of patient samples in a single run. To address strict requirements for control of assay parameters and output demanded by clinical diagnostics, we employ a control-based Precision-Recall and Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of several hundred patients performs with a specificity of 100% and sensitivity of 91% on samples with low viral loads, and a sensitivity of >95% on high viral loads associated with disease onset and peak transmissibility. This study establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , /patogenicidad , /genética , /virología , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificación de Ácido Nucleico , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Carga Viral
9.
Zool Res ; 42(2): 246-249, 2021 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-33709636

RESUMEN

Somatic mutations are a large category of genetic variations, which play an essential role in tumorigenesis. Detection of somatic single nucleotide variants (SNVs) could facilitate downstream analysis of tumorigenesis. Many computational methods have been developed to detect SNVs, but most require normal matched samples to differentiate somatic SNVs from the normal state, which can be difficult to obtain. Therefore, developing new approaches for detecting somatic SNVs without matched samples are crucial. In this work, we detected somatic mutations from individual tumor samples based on a novel machine learning approach, svmSomatic, using next-generation sequencing (NGS) data. In addition, as somatic SNV detection can be impacted by multiple mutations, with germline mutations and co-occurrence of copy number variations (CNVs) common in organisms, we used the novel approach to distinguish somatic and germline mutations based on the NGS data from individual tumor samples. In summary, svmSomatic: (1) considers the influence of CNV co-occurrence in detecting somatic mutations; and (2) trains a support vector machine algorithm to distinguish between somatic and germline mutations, without requiring normal matched samples. We further tested and compared svmSomatic with other common methods. Results showed that svmSomatic performance, as measured by F1-score, was significantly better than that of others using both simulation and real NGS data.


Asunto(s)
Aprendizaje Automático , Mutación/genética , Neoplasias/genética , Algoritmos , Animales , Biología Computacional/métodos , Variaciones en el Número de Copia de ADN , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias/metabolismo
10.
Nat Commun ; 12(1): 1605, 2021 03 11.
Artículo en Inglés | MEDLINE | ID: mdl-33707415

RESUMEN

Deep learning algorithms have been utilized to achieve enhanced performance in pattern-recognition tasks. The ability to learn complex patterns in data has tremendous implications in immunogenomics. T-cell receptor (TCR) sequencing assesses the diversity of the adaptive immune system and allows for modeling its sequence determinants of antigenicity. We present DeepTCR, a suite of unsupervised and supervised deep learning methods able to model highly complex TCR sequencing data by learning a joint representation of a TCR by its CDR3 sequences and V/D/J gene usage. We demonstrate the utility of deep learning to provide an improved 'featurization' of the TCR across multiple human and murine datasets, including improved classification of antigen-specific TCRs and extraction of antigen-specific TCRs from noisy single-cell RNA-Seq and T-cell culture-based assays. Our results highlight the flexibility and capacity for deep neural networks to extract meaningful information from complex immunogenomic data for both descriptive and predictive purposes.


Asunto(s)
Algoritmos , Aprendizaje Profundo , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Secuencia de Aminoácidos/genética , Animales , Bases de Datos Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Ratones , Redes Neurales de la Computación , RNA-Seq/métodos
11.
Nat Commun ; 12(1): 1361, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33649327

RESUMEN

Sperm contributes diverse RNAs to the zygote. While sperm small RNAs have been shown to impact offspring phenotypes, our knowledge of the sperm transcriptome, especially the composition of long RNAs, has been limited by the lack of sensitive, high-throughput experimental techniques that can distinguish intact RNAs from fragmented RNAs, known to abound in sperm. Here, we integrate single-molecule long-read sequencing with short-read sequencing to detect sperm intact RNAs (spiRNAs). We identify 3440 spiRNA species in mice and 4100 in humans. The spiRNA profile consists of both mRNAs and long non-coding RNAs, is evolutionarily conserved between mice and humans, and displays an enrichment in mRNAs encoding for ribosome. In sum, we characterize the landscape of intact long RNAs in sperm, paving the way for future studies on their biogenesis and functions. Our experimental and bioinformatics approaches can be applied to other tissues and organisms to detect intact transcripts.


Asunto(s)
Secuencia Conservada/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/genética , Imagen Individual de Molécula , Espermatozoides/metabolismo , Animales , Evolución Molecular , Ontología de Genes , Humanos , Masculino , Ratones Endogámicos C57BL , ARN/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Testículo/metabolismo , Transcriptoma/genética
12.
Nat Commun ; 12(1): 1652, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712618

RESUMEN

Annotation of polyadenylation sites from short-read RNA sequencing alone is a challenging computational task. Other algorithms rooted in DNA sequence predict potential polyadenylation sites; however, in vivo expression of a particular site varies based on a myriad of conditions. Here, we introduce aptardi (alternative polyadenylation transcriptome analysis from RNA-Seq data and DNA sequence information), which leverages both DNA sequence and RNA sequencing in a machine learning paradigm to predict expressed polyadenylation sites. Specifically, as input aptardi takes DNA nucleotide sequence, genome-aligned RNA-Seq data, and an initial transcriptome. The program evaluates these initial transcripts to identify expressed polyadenylation sites in the biological sample and refines transcript 3'-ends accordingly. The average precision of the aptardi model is twice that of a standard transcriptome assembler. In particular, the recall of the aptardi model (the proportion of true polyadenylation sites detected by the algorithm) is improved by over three-fold. Also, the model-trained using the Human Brain Reference RNA commercial standard-performs well when applied to RNA-sequencing samples from different tissues and different mammalian species. Finally, aptardi's input is simple to compile and its output is easily amenable to downstream analyses such as quantitation and differential expression.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Poliadenilación , Transcriptoma , Animales , Secuencia de Bases , Perfilación de la Expresión Génica , Humanos , ARN/química , ARN/metabolismo , Análisis de Secuencia de ARN , Biología de Sistemas
13.
Front Immunol ; 12: 641900, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33732261

RESUMEN

Human leukocyte antigen (HLA) class I molecules play a crucial role in the development of a specific immune response to viral infections by presenting viral peptides at the cell surface where they will be further recognized by T cells. In the present manuscript, we explored whether HLA class I genotypes can be associated with the critical course of Coronavirus Disease-19 by searching possible connections between genotypes of deceased patients and their age at death. HLA-A, HLA-B, and HLA-C genotypes of n = 111 deceased patients with COVID-19 (Moscow, Russia) and n = 428 volunteers were identified with next-generation sequencing. Deceased patients were split into two groups according to age at the time of death: n = 26 adult patients aged below 60 and n = 85 elderly patients over 60. With the use of HLA class I genotypes, we developed a risk score (RS) which was associated with the ability to present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) peptides by the HLA class I molecule set of an individual. The resulting RS was significantly higher in the group of deceased adults compared to elderly adults [p = 0.00348, area under the receiver operating characteristic curve (AUC ROC = 0.68)]. In particular, presence of HLA-A*01:01 allele was associated with high risk, while HLA-A*02:01 and HLA-A*03:01 mainly contributed to low risk. The analysis of patients with homozygosity strongly highlighted these results: homozygosity by HLA-A*01:01 accompanied early deaths, while only one HLA-A*02:01 homozygote died before 60 years of age. Application of the constructed RS model to an independent Spanish patients cohort (n = 45) revealed that the score was also associated with the severity of the disease. The obtained results suggest the important role of HLA class I peptide presentation in the development of a specific immune response to COVID-19.


Asunto(s)
/inmunología , Genotipo , Antígenos HLA-A/genética , Índice de Severidad de la Enfermedad , Factores de Edad , Anciano , Anciano de 80 o más Años , Alelos , /virología , Estudios de Cohortes , Femenino , Frecuencia de los Genes , Pruebas Genéticas/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
16.
Nat Protoc ; 16(3): 1511-1547, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33547443

RESUMEN

The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR-Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR-Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks.


Asunto(s)
Sistemas CRISPR-Cas/fisiología , Edición Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Animales , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , ADN/genética , Humanos , Motivos de Nucleótidos/genética , ARN Guia/genética , Proyectos de Investigación
17.
Methods Mol Biol ; 2243: 27-58, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606251

RESUMEN

The next-generation sequencing (NGS) technology has revolutionized research in genetics and genomics, resulting in massive NGS data and opening more fronts to answer unresolved issues in genetics. NGS data are usually stored at three levels: image files, sequence tags, and alignment reads. The sizes of these types of data usually range from several hundreds of gigabytes to several terabytes. Biostatisticians and bioinformaticians are typically working with the aligned NGS read count data (hence the last level of NGS data) for data modeling and interpretation.To horn in on the use of NGS technology, researchers utilize it to profile the whole genome to study DNA copy number variations (CNVs) for an individual subject (or patient) as well as groups of subjects (or patients). The resulting aligned NGS read count data are then modeled by proper mathematical and statistical approaches so that the loci of CNVs can be accurately detected. In this book chapter, a summary of most popularly used statistical methods for detecting CNVs using NGS data is given. The goal is to provide readers with a comprehensive resource of available statistical approaches for inferring DNA copy number variations using NGS data.


Asunto(s)
Variaciones en el Número de Copia de ADN/genética , Genoma Humano/genética , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos
18.
Methods Mol Biol ; 2243: 95-105, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606254

RESUMEN

High-throughput sequencing machines can read millions of DNA molecules in parallel in a short time and at a relatively low cost. As a consequence, researchers have access to databases with millions of genomic samples. Searching and analyzing these large amounts of data require efficient algorithms.Universal hitting sets are sets of words that must be present in any long enough string. Using small universal hitting sets, it is possible to increase the efficiency of many high-throughput sequencing data analyses. But, generating minimum-size universal hitting sets is a hard problem. In this chapter, we cover our algorithmic developments to produce compact universal hitting sets and some of their potential applications.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Algoritmos , ADN/genética , Humanos , Programas Informáticos
19.
Methods Mol Biol ; 2243: 81-94, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606253

RESUMEN

Advances in next generation sequencing (NGS) technologies resulted in a broad array of large-scale gene expression studies and an unprecedented volume of whole messenger RNA (mRNA) sequencing data, or the transcriptome (also known as RNA sequencing, or RNA-seq). These include the Genotype Tissue Expression project (GTEx) and The Cancer Genome Atlas (TCGA), among others. Here we cover some of the commonly used datasets, provide an overview on how to begin the analysis pipeline, and how to explore and interpret the data provided by these publicly available resources.


Asunto(s)
RNA-Seq/métodos , Análisis de Secuencia de ARN/métodos , Bases de Datos Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , ARN Mensajero/genética , Transcriptoma/genética
20.
Methods Mol Biol ; 2243: 123-141, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33606256

RESUMEN

In this chapter, we will present an outline of a typical experimental and bioinformatic workflow for identification of bacterial amplicon sequence variants (ASVs) present in a set of samples. This chapter is written from a bioinformatic point of view; therefore, the specific experimental protocols are not detailed, but rather the impact of various experimental decisions on the downstream analysis is described. Emphasis is made on the transition from reads to ASVs, describing the Deblur algorithm.


Asunto(s)
Variación Genética/genética , Microbiota/genética , ARN Ribosómico 16S/genética , Algoritmos , Animales , Bacterias/genética , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Metagenoma/genética , Flujo de Trabajo
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