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1.
Methods Mol Biol ; 2212: 277-289, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33733362

RESUMO

We report a step-by-step protocol to use pysster, a TensorFlow-based package for building deep neural networks on a broad range of epistatic sequences such as DNA, RNA, or annotated secondary structure sequences. Pysster provides users comprehensive supports for developing, training, and evaluating the self-defined deep neural networks on sequence data. Moreover, pysster allows users to easily visualize the resulting perditions, which is helpful to uncover the "black box" of deep neural networks. Here, we describe a step-by-step application of pysster to classify the RNA A-to-I editing regions and interpret the model predictions. To further demonstrate the generalizability of pysster, we utilized it to build and evaluated a new deep neural network on an artificial epistatic sequence dataset.


Assuntos
Aprendizado Profundo , Epistasia Genética , Modelos Genéticos , RNA/genética , Software , Sequência de Bases , Conjuntos de Dados como Assunto , Humanos , Edição de RNA , Curva ROC , Análise de Sequência/estatística & dados numéricos
2.
Nat Commun ; 12(1): 1654, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33712600

RESUMO

ADAR1 is involved in adenosine-to-inosine RNA editing. The cytoplasmic ADAR1p150 edits 3'UTR double-stranded RNAs and thereby suppresses induction of interferons. Loss of this ADAR1p150 function underlies the embryonic lethality of Adar1 null mice, pathogenesis of the severe autoimmune disease Aicardi-Goutières syndrome, and the resistance developed in cancers to immune checkpoint blockade. In contrast, the biological functions of the nuclear-localized ADAR1p110 remain largely unknown. Here, we report that ADAR1p110 regulates R-loop formation and genome stability at telomeres in cancer cells carrying non-canonical variants of telomeric repeats. ADAR1p110 edits the A-C mismatches within RNA:DNA hybrids formed between canonical and non-canonical variant repeats. Editing of A-C mismatches to I:C matched pairs facilitates resolution of telomeric R-loops by RNase H2. This ADAR1p110-dependent control of telomeric R-loops is required for continued proliferation of telomerase-reactivated cancer cells, revealing the pro-oncogenic nature of ADAR1p110 and identifying ADAR1 as a promising therapeutic target of telomerase positive cancers.


Assuntos
Adenosina Desaminase/metabolismo , Instabilidade Genômica , Neoplasias/metabolismo , Estruturas R-Loop , Edição de RNA , Proteínas de Ligação a RNA/metabolismo , Telômero/metabolismo , Adenosina Desaminase/genética , Animais , Linhagem Celular Tumoral , DNA , Dano ao DNA , Genômica , Células HEK293 , Células HeLa , Humanos , Camundongos , Neoplasias/genética , Proteínas de Ligação a RNA/genética , Transcriptoma
3.
Virology ; 556: 62-72, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33545556

RESUMO

Members of the APOBEC family of cytidine deaminases show antiviral activities in mammalian cells through lethal editing in the genomes of small DNA viruses, herpesviruses and retroviruses, and potentially those of RNA viruses such as coronaviruses. Consistent with the latter, APOBEC-like directional C→U transitions of genomic plus-strand RNA are greatly overrepresented in SARS-CoV-2 genome sequences of variants emerging during the COVID-19 pandemic. A C→U mutational process may leave evolutionary imprints on coronavirus genomes, including extensive homoplasy from editing and reversion at targeted sites and the occurrence of driven amino acid sequence changes in viral proteins. If sustained over longer periods, this process may account for the previously reported marked global depletion of C and excess of U bases in human seasonal coronavirus genomes. This review synthesizes the current knowledge on APOBEC evolution and function and the evidence of their role in APOBEC-mediated genome editing of SARS-CoV-2 and other coronaviruses.


Assuntos
Desaminases APOBEC/metabolismo , Coronavirus/genética , Evolução Molecular , Genoma Viral/genética , Edição de RNA , Desaminases APOBEC/química , Desaminases APOBEC/genética , Animais , Infecções por Coronavirus/virologia , Humanos , Mutação , /genética
4.
Int J Mol Sci ; 22(2)2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33440692

RESUMO

The early vascular plants in the genus Selaginella, which is the sole genus of the Selaginellaceae family, have an important place in evolutionary history, along with ferns, as such plants are valuable resources for deciphering plant evolution. In this study, we sequenced and assembled the plastid genome (plastome) sequences of two Selaginella tamariscina individuals, as well as Selaginella stauntoniana and Selaginella involvens. Unlike the inverted repeat (IR) structures typically found in plant plastomes, Selaginella species had direct repeat (DR) structures, which were confirmed by Oxford Nanopore long-read sequence assembly. Comparative analyses of 19 lycophytes, including two Huperzia and one Isoetes species, revealed unique phylogenetic relationships between Selaginella species and related lycophytes, reflected by structural rearrangements involving two rounds of large inversions that resulted in dynamic changes between IR and DR blocks in the plastome sequence. Furthermore, we present other uncommon characteristics, including a small genome size, drastic reductions in gene and intron numbers, a high GC content, and extensive RNA editing. Although the 16 Selaginella species examined may not fully represent the genus, our findings suggest that Selaginella plastomes have undergone unique evolutionary events yielding genomic features unparalleled in other lycophytes, ferns, or seed plants.


Assuntos
Genoma de Planta , Genomas de Plastídeos , Genômica , Selaginellaceae/genética , Composição de Bases , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Tamanho do Genoma , Genômica/métodos , Íntrons , Filogenia , Edição de RNA , Selaginellaceae/classificação
6.
Int J Mol Sci ; 22(2)2021 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-33430133

RESUMO

Currently, for seemingly every type of cancer, dysregulated levels of non-coding RNAs (ncRNAs) are reported and non-coding transcripts are expected to be the next class of diagnostic and therapeutic tools in oncology. Recently, alterations to the ncRNAs transcriptome have emerged as a novel hallmark of cancer. Historically, ncRNAs were characterized mainly as regulators and little attention was paid to the mechanisms that regulate them. The role of modifications, which can control the function of ncRNAs post-transcriptionally, only recently began to emerge. Typically, these modifications can be divided into reversible (i.e., chemical modifications: m5C, hm5C, m6A, m1A, and pseudouridine) and non-reversible (i.e., editing: ADAR dependent, APOBEC dependent and ADAR/APOBEC independent). The first research papers showed that levels of these modifications are altered in cancer and can be part of the tumorigenic process. Hence, the aim of this review paper is to describe the most common regulatory modifications (editing and chemical modifications) of the traditionally considered "non-functional" ncRNAs (i.e., microRNAs, long non-coding RNAs and circular RNAs) in the context of malignant disease. We consider that only by understanding this extra regulatory layer is it possible to translate the knowledge about ncRNAs and their modifications into clinical practice.


Assuntos
Neoplasias/genética , Edição de RNA/genética , RNA não Traduzido/genética , Transcriptoma/genética , Carcinogênese/genética , Humanos , Neoplasias/terapia , RNA não Traduzido/uso terapêutico
7.
Methods Mol Biol ; 2162: 115-152, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32926381

RESUMO

CRISPR-Display uses the S. pyogenes Cas9 protein to posttranscriptionally localize noncoding RNA (ncRNA) domains to any genomic site, by directly fusing these domains to the Cas9 sgRNA cofactor. This versatile technology enables numerous applications for interrogating natural chromatin-regulatory ncRNAs, or for utilizing artificial ncRNA and ribonucleoprotein (RNP) devices at individual chromatin loci. To achieve these, a successful CRISPR-Display experiment requires that chimeric sgRNA-ncRNA fusions are stably expressed and incorporated into Cas9 complexes, and that they retain their ncRNA "cargo" domains at the targeted genomic sites. Here, I describe a workflow for designing, building, and testing such chimeric sgRNA-ncRNA expression constructs. I detail strategies for choosing expression systems and sgRNA insertion topologies, for assaying the incorporation of sgRNA-ncRNA fusions into functional Cas9 complexes, and for surveying the activities of ncRNA domains at targeted genomic loci. This establishes an initial set of "best practices" for the design and implementation of CRISPR-Display experiments.


Assuntos
Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Genômica/métodos , RNA Guia/genética , Proteína 9 Associada à CRISPR/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Edição de RNA/genética , RNA não Traduzido/genética , Ribonucleoproteínas/genética
8.
Methods Mol Biol ; 2181: 13-34, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729072

RESUMO

Computers are able to systematically exploit RNA-seq data allowing us to efficiently detect RNA editing sites in a genome-wide scale. This chapter introduces a very flexible computational framework for detecting RNA editing sites in plant organelles. This framework comprises three major steps: RNA-seq data processing, RNA read alignment, and RNA editing site detection. Each step is discussed in sufficient detail to be implemented by the reader. As a study case, the framework will be used with publicly available sequencing data to detect C-to-U RNA editing sites in the coding sequences of the mitochondrial genome of Nicotiana tabacum.


Assuntos
Biologia Computacional/métodos , Genoma Mitocondrial , Mitocôndrias/genética , Edição de RNA/genética , RNA Mitocondrial/genética , Tabaco/genética , Citidina/química , Citidina/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mitocôndrias/metabolismo , RNA Mitocondrial/metabolismo , Software , Tabaco/metabolismo , Transcriptoma , Uridina/química , Uridina/genética
9.
Methods Mol Biol ; 2181: 1-12, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729071

RESUMO

RNA editing by cytidine (C) to uridine (U) conversions frequently occurs in land plant mitochondria and plastids. Target cytidines are specifically recognized by nuclear-encoded pentatricopeptide repeat (PPR) proteins in a sequence-specific manner. In the moss Physcomitrella patens, all PPR editing factors possess the DYW-deaminase domain at the C-terminus. Here, we describe methods for the direct sequencing of cDNA to detect RNA editing events and the RNA electrophoresis mobility shift assay (REMSA) to analyze the specific binding of PPR editing factors to their target RNA.


Assuntos
Bryopsida/genética , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Mitocôndrias/genética , Proteínas de Plantas/genética , Plastídeos/genética , Edição de RNA/genética , RNA de Plantas/genética , Bryopsida/metabolismo , Citidina/química , Citidina/genética , DNA Complementar/genética , Mitocôndrias/metabolismo , Proteínas de Plantas/metabolismo , Plastídeos/metabolismo , RNA de Plantas/metabolismo , Uridina/química , Uridina/genética
10.
Methods Mol Biol ; 2181: 35-50, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729073

RESUMO

RNA editing is an important posttranscriptional process that alters the genetic information of RNA encoded by genomic DNA. Adenosine-to-inosine (A-to-I) editing is the most prevalent type of RNA editing in animal kingdom, catalyzed by adenosine deaminases acting on RNA (ADARs). Recently, genome-wide A-to-I RNA editing is discovered in fungi, involving adenosine deamination mechanisms distinct from animals. Aiming to draw more attention to RNA editing in fungi, here we discuss the considerations for deep sequencing data preparation and the available various methods for detecting RNA editing, with a special emphasis on their usability for fungal RNA editing detection. We describe computational protocols for the identification of candidate RNA editing sites in fungi by using two software packages REDItools and RES-Scanner with RNA sequencing (RNA-Seq) and genomic DNA sequencing (DNA-Seq) data.


Assuntos
Biologia Computacional/métodos , Fungos/genética , Genoma Fúngico , Edição de RNA/genética , RNA Fúngico/genética , Software , Adenosina/química , Adenosina/genética , Fungos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Inosina/química , Inosina/genética , RNA Fúngico/metabolismo
11.
Methods Mol Biol ; 2181: 51-67, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729074

RESUMO

The AID/APOBEC family of enzymes are cytidine deaminases that act upon DNA and RNA. Among APOBECs, the best characterized family member to act on RNA is the enzyme APOBEC1. APOBEC1-mediated RNA editing plays a key role in lipid metabolism and in maintenance of brain homeostasis. Editing can be easily detected in RNA-seq data as a cytosine to thymine (C-to-T) change with regard to the reference. However, there are many other sources of base conversions relative to reference, such as PCR errors, SNPs, and even DNA editing by mutator APOBECs. Furthermore, APOBEC1 exhibits disparate activity in different cell types, with respect to which transcripts are edited and the level to which they are edited. When considering these potential sources of error and variability, an RNA-seq comparison between wild-type APOBEC1 sample and a matched control with an APOBEC1 knockout is a reliable method for the discrimination of true sites edited by APOBEC1. Here we present a detailed description of a method for studying APOBEC1 RNA editing, specifically in the murine macrophage cell line RAW 264.7. Our method covers the production of an APOBEC1 knockout cell line using the CRISPR/Cas9 system, through to experimental validation and quantification of editing sites (where we discuss a recently published algorithm (termed MultiEditR) which allows for the detection and quantification of RNA editing from Sanger sequencing). Importantly, this same protocol can be adapted to any RNA modification detectable by RNA-seq analysis for which the responsible protein is known.


Assuntos
Desaminase APOBEC-1/genética , Sistemas CRISPR-Cas , Biologia Computacional/métodos , Citidina/genética , Macrófagos/metabolismo , Edição de RNA/genética , Uridina/genética , Desaminase APOBEC-1/antagonistas & inibidores , Animais , Citidina/química , Sequenciamento de Nucleotídeos em Larga Escala , Macrófagos/citologia , Camundongos , Células RAW 264.7 , RNA Mensageiro/genética , Uridina/química
12.
Methods Mol Biol ; 2181: 69-81, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729075

RESUMO

APOBEC1 is a member of the AID/APOBECs, a group of deaminases responsible for the editing of C>U in both DNA and RNA. APOBEC1 is physiologically involved in C>U RNA editing: while hundreds of targets have been discovered in mice, in humans the only well-characterized target of APOBEC1 is the apolipoprotein B (ApoB) transcript. APOBEC1 edits a CAA codon into a stop codon, which causes the translation of a truncated form of ApoB. A number of assays have been developed to investigate this process. Early assays, poisoned primer extension and Sanger sequencing, have focused on accuracy and sensitivity but rely on extraction of the RNA from tissues and cells. More recently, the need to visualize the RNA editing process directly in live cells have led to the development of fluorescence-based tools. These assays detect RNA editing through reporters whose editing causes a change in cellular localization or a change in fluorescent properties. Here we review the available assays to quantify RNA editing, and we present the protocol for cytofluorimetric analysis using a double-fluorescent reporter.


Assuntos
Desaminase APOBEC-1/genética , Biologia Computacional/métodos , Citidina/genética , Edição de RNA/genética , RNA Mensageiro/genética , Frações Subcelulares/metabolismo , Uridina/genética , Desaminase APOBEC-1/metabolismo , Citidina/química , Genes Reporter , Células HEK293 , Células Hep G2 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Mensageiro/metabolismo , Uridina/química
13.
Methods Mol Biol ; 2181: 83-95, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729076

RESUMO

RNA is subjected to over 100 different types of chemical modifications inside the cell. These modifications have various effects on its function and expression, resulting in RNA diversity. RNA editing or conversion of adenosine to inosine (A-to-I) in a double-stranded RNA is a type of RNA modification that can introduce mutations into the precursor of microRNA (miRNA). It can also regulate miRNA processing independently of A-to-I RNA editing. This chapter outlines the role of an A-to-I RNA editing enzyme ADAR in miRNA processing and the experimental systems used to analyze the interaction between miRNAs and ADAR.


Assuntos
Adenosina Desaminase/metabolismo , Adenosina/genética , Inosina/genética , MicroRNAs/metabolismo , Edição de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Adenosina/química , Adenosina Desaminase/genética , Humanos , Inosina/química , MicroRNAs/genética , Interferência de RNA , Proteínas de Ligação a RNA/genética
14.
Methods Mol Biol ; 2181: 97-111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729077

RESUMO

The conversion of adenosine to inosine (A to I) by RNA editing represents a common posttranscriptional mechanism for diversification of both the transcriptome and proteome, and is a part of the cellular response for innate immune tolerance. Due to its preferential base-pairing with cytosine (C), inosine (I) is recognized as guanosine (G) by reverse transcriptase, as well as the cellular splicing and translation machinery. A-to-I editing events appear as A-G discrepancies between genomic DNA and cDNA sequences. Molecular analyses of RNA editing have leveraged these nucleoside differences to quantify RNA editing in ensemble populations of RNA transcripts and within individual cDNAs using high-throughput sequencing or Sanger sequencing-derived analysis of electropherogram peak heights. Here, we briefly review and compare these methods of RNA editing quantification, as well as provide experimental protocols by which such analyses may be achieved.


Assuntos
Adenosina/análise , DNA Complementar/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inosina/análise , Edição de RNA/genética , Transcriptoma , Adenosina/genética , DNA Complementar/genética , Genoma Humano , Humanos , Inosina/genética
15.
Methods Mol Biol ; 2181: 113-148, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729078

RESUMO

RNA editing of adenosines to inosines contributes to a wide range of biological processes by regulating gene expression post-transcriptionally. To understand the effect, accurate mapping of inosines is necessary. The most conventional method to identify an editing site is to compare the cDNA sequence with its corresponding genomic sequence. However, this method has a high false discovery rate because guanosine signals, due to experimental errors or noise in the obtained sequences, contaminate genuine inosine signals detected as guanosine. To ensure high accuracy, we developed the Inosine Chemical Erasing (ICE) method to accurately and biochemically identify inosines in RNA strands utilizing inosine cyanoethylation and reverse transcription-PCR. Furthermore, we applied this technique to next-generation sequencing technology, called ICE-seq, to conduct an unbiased genome-wide screening of A-to-I editing sites in the transcriptome.


Assuntos
Adenosina/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inosina/análise , Edição de RNA/genética , Transcriptoma , Adenosina/genética , Genoma Humano , Humanos , Inosina/genética
16.
Methods Mol Biol ; 2181: 149-162, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729079

RESUMO

Alu elements are repetitive short interspersed elements prevalent in the primate genome. These repeats account for over 10% of the genome with more than a million highly similar copies. A direct outcome of this is an enrichment in long structures of stable dsRNA, which are the target of adenosine deaminases acting on RNAs (ADARs), the enzymes catalyzing A-to-I RNA editing. Indeed, A-to-I editing by ADARs is extremely abundant in primates: over a hundred million editing sites exist in their genomes. However, despite the radical increase in ADAR targets brought on by the introduction of Alu elements, the few evolutionary conserved editing sites manage to retain their editing levels. Here, we review and discuss the cost of having an unusual amount of dsRNA and editing in the transcriptome, as well as the opportunities it presents, which possibly contributed to accelerating primate evolution.


Assuntos
Adenosina Desaminase/metabolismo , Adenosina/análise , Elementos Alu , Inosina/análise , Edição de RNA/genética , RNA de Cadeia Dupla , Transcriptoma , Adenosina/genética , Adenosina Desaminase/genética , Animais , Humanos , Inosina/genética , Primatas
17.
Methods Mol Biol ; 2181: 163-176, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729080

RESUMO

Adenosine-to-inosine (A-to-I) RNA editing is a fundamental posttranscriptional mechanism that greatly diversifies the transcriptome in many living organisms, including mammals. Multiple studies have demonstrated the importance of this process not just in normal development and physiology but also in various human diseases. Importantly, the precise editing level of a site may have downstream consequences on cellular behavior. Hence, the editing levels should be quantified as accurately as possible. In this chapter, we describe how to examine RNA editing in human and mouse tissues. The rapid development of next-generation sequencing technologies is affording us an unprecedented ability to accurately measure the editing levels of numerous sites simultaneously. Our experimental workflow includes the harvesting of high-quality RNA samples and the construction of different high-throughput sequencing libraries. We also delineate the computational steps needed to analyze the sequencing data from an Illumina platform.


Assuntos
Adenosina/análise , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inosina/análise , Edição de RNA/genética , RNA/genética , Transcriptoma , Adenosina/genética , Animais , Genoma , Humanos , Inosina/genética , Camundongos
18.
Methods Mol Biol ; 2170: 1-18, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32797447

RESUMO

Application of the CRISPR-Cas prokaryotic immune system for single-stranded RNA targeting will have significant impacts on RNA analysis and engineering. The class 2 Type VI CRISPR-Cas13 system is an RNA-guided RNA-nuclease system capable of binding and cleaving target single-stranded RNA substrates in a sequence-specific manner. In addition to RNA interference, the Cas13a system has application from manipulating RNA modifications, to editing RNA sequence, to use as a nucleic acid detection tool. This protocol uses the Cas13a ortholog from Leptotrichia buccalis for transient expression in plant cells providing antiviral defense. We cover all the necessary information for cloning the Cas13 protein, crRNA guide cassette, performing transient Agrobacterium-mediated expression of the necessary Cas13a components and target RNA-virus, visualization of virus infection, and molecular quantification of viral accumulation using quantitative PCR.


Assuntos
Biotecnologia/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de RNA/genética , Tabaco/metabolismo , Interferência de RNA/fisiologia , Tabaco/genética , Transcriptoma/genética
19.
Methods Mol Biol ; 2181: 177-191, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729081

RESUMO

RNA editing is a widespread co/posttranscriptional mechanism affecting primary RNAs by specific nucleotide modifications, which plays relevant roles in molecular processes including regulation of gene expression and/or processing of noncoding RNAs (ncRNAs). In recent years, the detection of editing sites has been greatly improved through the availability of high-throughput RNA sequencing technologies. Several pipelines, employing various read mappers and variant callers with a wide range of adjustable parameters, are currently available for the detection of RNA editing events. Hereafter, we describe some of the most recent and popular tools and provide guidelines for the detection of RNA editing in massive transcriptome data.


Assuntos
Biologia Computacional/métodos , Edição de RNA/fisiologia , Animais , Biologia Computacional/normas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Guias de Prática Clínica como Assunto , Análise de Sequência de RNA , Transcriptoma
20.
Methods Mol Biol ; 2181: 193-212, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32729082

RESUMO

The advent of deep sequencing technologies has greatly improved the study of complex eukaryotic genomes and transcriptomes, allowing the investigation of posttranscriptional molecular mechanisms as alternative splicing and RNA editing at unprecedented throughput and resolution. The most prevalent type of RNA editing in higher eukaryotes is the deamination of adenosine to inosine (A-to-I) in double-stranded RNAs. Depending on the RNA type or the RNA region involved, A-to-I RNA editing contributes to the transcriptome and proteome diversity.Hereafter, we present an easy and reproducible computational protocol for the identification of candidate RNA editing sites in humans using deep transcriptome (RNA-Seq) and genome (DNA-Seq) sequencing.


Assuntos
DNA/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Edição de RNA/fisiologia , RNA/análise , Animais , Biologia Computacional/instrumentação , Biologia Computacional/métodos , Computadores , Sequenciamento de Nucleotídeos em Larga Escala/instrumentação , Humanos , Análise de Sequência de RNA/métodos , Software , Transcriptoma/fisiologia
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