RESUMO
SQANTI3 is a tool designed for the quality control, curation and annotation of long-read transcript models obtained with third-generation sequencing technologies. Leveraging its annotation framework, SQANTI3 calculates quality descriptors of transcript models, junctions and transcript ends. With this information, potential artifacts can be identified and replaced with reliable sequences. Furthermore, the integrated functional annotation feature enables subsequent functional iso-transcriptomics analyses.
Assuntos
Anotação de Sequência Molecular , Transcriptoma , Humanos , Anotação de Sequência Molecular/métodos , Software , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Isoformas de Proteínas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Advances in affordable transcriptome sequencing combined with better exon and gene prediction has motivated many to compare transcription across the tree of life. We develop a mathematical framework to calculate complexity and compare transcript models. Structural features, i.e. intron retention (IR), donor/acceptor site variation, alternative exon cassettes, alternative 5'/3' UTRs, are compared and the distance between transcript models is calculated with nucleotide level precision. All metrics are implemented in a PyPi package, TranD and output can be used to summarize splicing patterns for a transcriptome (1GTF) and between transcriptomes (2GTF). TranD output enables quantitative comparisons between: annotations augmented by empirical RNA-seq data and the original transcript models; transcript model prediction tools for longread RNA-seq (e.g. FLAIR versus Isoseq3); alternate annotations for a species (e.g. RefSeq vs Ensembl); and between closely related species. In C. elegans, Z. mays, D. melanogaster, D. simulans and H. sapiens, alternative exons were observed more frequently in combination with an alternative donor/acceptor than alone. Transcript models in RefSeq and Ensembl are linked and both have unique transcript models with empirical support. D. melanogaster and D. simulans, share many transcript models and long-read RNAseq data suggests that both species are under-annotated. We recommend combined references.
Assuntos
Processamento Alternativo , Transcriptoma , Animais , Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Nucleotídeos , Splicing de RNA , Análise de Sequência de RNA , Especificidade da Espécie , Transcriptoma/genética , SoftwareRESUMO
In Drosophila melanogaster and D. simulans head tissue, 60% of orthologous genes show evidence of sex-biased expression in at least one species. Of these, â¼39% (2,192) are conserved in direction. We hypothesize enrichment of open chromatin in the sex where we see expression bias and closed chromatin in the opposite sex. Male-biased orthologs are significantly enriched for H3K4me3 marks in males of both species (â¼89% of male-biased orthologs vs. â¼76% of unbiased orthologs). Similarly, female-biased orthologs are significantly enriched for H3K4me3 marks in females of both species (â¼90% of female-biased orthologs vs. â¼73% of unbiased orthologs). The sex-bias ratio in female-biased orthologs was similar in magnitude between the two species, regardless of the closed chromatin (H3K27me2me3) marks in males. However, in male-biased orthologs, the presence of H3K27me2me3 in both species significantly reduced the correlation between D. melanogaster sex-bias ratio and the D. simulans sex-bias ratio. Male-biased orthologs are enriched for evidence of positive selection in the D. melanogaster group. There are more male-biased genes than female-biased genes in both species. For orthologs with gains/losses of sex-bias between the two species, there is an excess of male-bias compared to female-bias, but there is no consistent pattern in the relationship between H3K4me3 or H3K27me2me3 chromatin marks and expression. These data suggest chromatin state is a component of the maintenance of sex-biased expression and divergence of sex-bias between species is reflected in the complexity of the chromatin status.
Assuntos
Cromatina , Drosophila melanogaster , Animais , Feminino , Masculino , Drosophila melanogaster/genética , Cromatina/genética , Drosophila simulans/genética , Evolução Molecular , Drosophila/genéticaRESUMO
BACKGROUND: Genomic complexity is a growing field of evolution, with case studies for comparative evolutionary analyses in model and emerging non-model systems. Understanding complexity and the functional components of the genome is an untapped wealth of knowledge ripe for exploration. With the "remarkable lack of correspondence" between genome size and complexity, there needs to be a way to quantify complexity across organisms. In this study, we use a set of complexity metrics that allow for evaluating changes in complexity using TranD. RESULTS: We ascertain if complexity is increasing or decreasing across transcriptomes and at what structural level, as complexity varies. In this study, we define three metrics - TpG, EpT, and EpG- to quantify the transcriptome's complexity that encapsulates the dynamics of alternative splicing. Here we compare complexity metrics across 1) whole genome annotations, 2) a filtered subset of orthologs, and 3) novel genes to elucidate the impacts of orthologs and novel genes in transcript model analysis. Effective Exon Number (EEN) issued to compare the distribution of exon sizes within transcripts against random expectations of uniform exon placement. EEN accounts for differences in exon size, which is important because novel gene differences in complexity for orthologs and whole-transcriptome analyses are biased towards low-complexity genes with few exons and few alternative transcripts. CONCLUSIONS: With our metric analyses, we are able to quantify changes in complexity across diverse lineages with greater precision and accuracy than previous cross-species comparisons under ortholog conditioning. These analyses represent a step toward whole-transcriptome analysis in the emerging field of non-model evolutionary genomics, with key insights for evolutionary inference of complexity changes on deep timescales across the tree of life. We suggest a means to quantify biases generated in ortholog calling and correct complexity analysis for lineage-specific effects. With these metrics, we directly assay the quantitative properties of newly formed lineage-specific genes as they lower complexity.
Assuntos
Eucariotos , Transcriptoma , Eucariotos/genética , Genômica , Perfilação da Expressão Gênica , Genoma , Processamento Alternativo , Evolução MolecularRESUMO
Influenza A virus (IAV) genomes are composed of eight single-stranded RNA segments that are coated by viral nucleoprotein (NP) molecules. Classically, the interaction between NP and viral RNA (vRNA) is depicted as a uniform pattern of 'beads on a string'. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP), we identified the vRNA binding profiles of NP for two H1N1 IAV strains in virions. Contrary to the prevailing model for vRNA packaging, NP does not bind vRNA uniformly in the A/WSN/1933 and A/California/07/2009 strains, but instead each vRNA segment exhibits a unique binding profile, containing sites that are enriched or poor in NP association. Intriguingly, both H1N1 strains have similar yet distinct NP binding profiles despite extensive sequence conservation. Peaks identified by HITS-CLIP were verified as true NP binding sites based on insensitivity to DNA antisense oligonucleotide-mediated RNase H digestion. Moreover, nucleotide content analysis of NP peaks revealed that these sites are relatively G-rich and U-poor compared to the genome-wide nucleotide content, indicating an as-yet unidentified sequence bias for NP association in vivo. Taken together, our genome-wide study of NP-vRNA interaction has implications for the understanding of influenza vRNA architecture and genome packaging.
Assuntos
Genoma Viral , Vírus da Influenza A Subtipo H1N1/genética , Nucleoproteínas/química , RNA Viral/química , Proteínas Virais/química , Vírion/genética , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Vírus da Influenza A Subtipo H1N1/metabolismo , Vírus da Influenza A Subtipo H1N1/ultraestrutura , Modelos Moleculares , Nucleoproteínas/genética , Nucleoproteínas/metabolismo , Oligonucleotídeos Antissenso/química , Ligação Proteica , RNA Viral/genética , RNA Viral/metabolismo , Ribonuclease H/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/metabolismo , Vírion/ultraestrutura , Montagem de Vírus/genéticaRESUMO
Epstein-Barr virus (EBV) expresses an abundant nuclear noncoding RNA called EBER2, which interacts with and acts as a guide RNA for the host transcription factor PAX5. This ribonucleoprotein complex localizes to the terminal repeat (TR) regions of the EBV genome via RNA-RNA interactions between EBER2 and nascent transcripts originating from these target sites. Given the fact that EBER2 base pairs with a viral RNA, we developed a protocol to identify EBER2-interacting RNAs in a transcriptome-wide manner. Our approach entails psoralen-mediated crosslinking, selection with antisense oligonucleotides targeting EBER2, and RNase V1 digestion coupled to next-generation sequencing. The use of RNase V1 circumvents the need of extensive computational analysis post data acquisition to search for predicted RNA hybrids, as the RNase V1 cleavage site marks the region of RNA duplex formation. As proof of principle, we show that our approach correctly identifies the known EBER2 interaction with TR RNAs. Moreover, we identify the host functional noncoding RNAs MRP, H1, and 7SL RNAs as well as three putative enhancer RNAs as candidate EBER2-interacting RNAs. As all of these gene loci exhibit PAX5 occupancy, we propose that EBER2 is recruited to these sites through its binding partner PAX5 and forms RNA-RNA interactions with nascent transcripts on chromatin. Thus, our novel approach facilitates the identification of targeted RNA-RNA-interactions and minimizes the need of downstream computational analyses to predict RNA duplexes.
Assuntos
Herpesvirus Humano 4/genética , Interações Hospedeiro-Patógeno/genética , RNA não Traduzido/metabolismo , RNA Viral/metabolismo , Endorribonucleases/genética , Endorribonucleases/metabolismo , Elementos Facilitadores Genéticos , Perfilação da Expressão Gênica , Herpesvirus Humano 4/patogenicidade , Humanos , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , RNA não Traduzido/genética , Ribonuclease P/genética , Sequências Repetidas TerminaisRESUMO
We propose a new model for the association of chromatin state and sex-bias in expression. We hypothesize enrichment of open chromatin in the sex where we see expression bias (OS) and closed chromatin in the opposite sex (CO). In this study of D. melanogaster and D. simulans head tissue, sex-bias in expression is associated with H3K4me3 (open mark) in males for male-biased genes and in females for female-biased genes in both species. Sex-bias in expression is also largely conserved in direction and magnitude between the two species on the X and autosomes. In male-biased orthologs, the sex-bias ratio is more divergent between species if both species have H3K27me2me3 marks in females compared to when either or neither species has H3K27me2me3 in females. H3K27me2me3 marks in females are associated with male-bias in expression on the autosomes in both species, but on the X only in D. melanogaster . In female-biased orthologs the relationship between the species for the sex-bias ratio is similar regardless of the H3K27me2me3 marks in males. Female-biased orthologs are more similar in the ratio of sex-bias than male-biased orthologs and there is an excess of male-bias in expression in orthologs that gain/lose sex-bias. There is an excess of male-bias in sex-limited expression in both species suggesting excess male-bias is due to rapid evolution between the species. The X chromosome has an enrichment in male-limited H3K4me3 in both species and an enrichment of sex-bias in expression compared to the autosomes.
RESUMO
The emergence of long-read RNA sequencing (lrRNA-seq) has provided an unprecedented opportunity to analyze transcriptomes at isoform resolution. However, the technology is not free from biases, and transcript models inferred from these data require quality control and curation. In this study, we introduce SQANTI3, a tool specifically designed to perform quality analysis on transcriptomes constructed using lrRNA-seq data. SQANTI3 provides an extensive naming framework to describe transcript model diversity in comparison to the reference transcriptome. Additionally, the tool incorporates a wide range of metrics to characterize various structural properties of transcript models, such as transcription start and end sites, splice junctions, and other structural features. These metrics can be utilized to filter out potential artifacts. Moreover, SQANTI3 includes a Rescue module that prevents the loss of known genes and transcripts exhibiting evidence of expression but displaying low-quality features. Lastly, SQANTI3 incorporates IsoAnnotLite, which enables functional annotation at the isoform level and facilitates functional iso-transcriptomics analyses. We demonstrate the versatility of SQANTI3 in analyzing different data types, isoform reconstruction pipelines, and sequencing platforms, and how it provides novel biological insights into isoform biology. The SQANTI3 software is available at https://github.com/ConesaLab/SQANTI3 .
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We examine the impact of sustained elevated ozone concentration on the leaf transcriptome of 5 diverse maize inbred genotypes, which vary in physiological sensitivity to ozone (B73, Mo17, Hp301, C123, and NC338), using long reads to assemble transcripts and short reads to quantify expression of these transcripts. More than 99% of the long reads, 99% of the assembled transcripts, and 97% of the short reads map to both B73 and Mo17 reference genomes. Approximately 95% of the genes with assembled transcripts belong to known B73-Mo17 syntenic loci and 94% of genes with assembled transcripts are present in all temperate lines in the nested association mapping pan-genome. While there is limited evidence for alternative splicing in response to ozone stress, there is a difference in the magnitude of differential expression among the 5 genotypes. The transcriptional response to sustained ozone stress in the ozone resistant B73 genotype (151 genes) was modest, while more than 3,300 genes were significantly differentially expressed in the more sensitive NC338 genotype. There is the potential for tandem duplication in 30% of genes with assembled transcripts, but there is no obvious association between potential tandem duplication and differential expression. Genes with a common response across the 5 genotypes (83 genes) were associated with photosynthesis, in particular photosystem I. The functional annotation of genes not differentially expressed in B73 but responsive in the other 4 genotypes (789) identifies reactive oxygen species. This suggests that B73 has a different response to long-term ozone exposure than the other 4 genotypes. The relative magnitude of the genotypic response to ozone, and the enrichment analyses are consistent regardless of whether aligning short reads to: long read assembled transcripts; the B73 reference; the Mo17 reference. We find that prolonged ozone exposure directly impacts the photosynthetic machinery of the leaf.
Assuntos
Ozônio , Zea mays , Regulação da Expressão Gênica de Plantas , Genótipo , Ozônio/metabolismo , Ozônio/toxicidade , Folhas de Planta/genética , Folhas de Planta/metabolismo , Transcriptoma , Zea mays/genética , Zea mays/metabolismoRESUMO
Sweet corn is one of the most important vegetables in the United States and Canada. Here, we present a de novo assembly of a sweet corn inbred line Ia453 with the mutated shrunken2-reference allele (Ia453-sh2). This mutation accumulates more sugar and is present in most commercial hybrids developed for the processing and fresh markets. The ten pseudochromosomes cover 92% of the total assembly and 99% of the estimated genome size, with a scaffold N50 of 222.2 Mb. This reference genome completely assembles the large structural variation that created the mutant sh2-R allele. Furthermore, comparative genomics analysis with six field corn genomes highlights differences in single-nucleotide polymorphisms, structural variations, and transposon composition. Phylogenetic analysis of 5,381 diverse maize and teosinte accessions reveals genetic relationships between sweet corn and other types of maize. Our results show evidence for a common origin in northern Mexico for modern sweet corn in the U.S. Finally, population genomic analysis identifies regions of the genome under selection and candidate genes associated with sweet corn traits, such as early flowering, endosperm composition, plant and tassel architecture, and kernel row number. Our study provides a high-quality reference-genome sequence to facilitate comparative genomics, functional studies, and genomic-assisted breeding for sweet corn.
Assuntos
Evolução Molecular , Genética Populacional , Genoma de Planta , Zea mays/genética , Alelos , Elementos de DNA Transponíveis/genética , Loci Gênicos , Haplótipos/genética , Anotação de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Análise de Sequência de DNA , Zea mays/anatomia & histologiaRESUMO
The genomes of influenza A and B viruses have eight, single-stranded RNA segments that exist in the form of a viral ribonucleoprotein complex in association with nucleoprotein (NP) and an RNA-dependent RNA polymerase complex. We previously used high-throughput RNA sequencing coupled with crosslinking immunoprecipitation (HITS-CLIP) to examine where NP binds to the viral RNA (vRNA) and demonstrated for two H1N1 strains that NP binds vRNA in a non-uniform, non-random manner. In this study, we expand on those initial observations and describe the NP-vRNA binding profile for a seasonal H3N2 and influenza B virus. We show that, similar to H1N1 strains, NP binds vRNA in a non-uniform and non-random manner. Each viral gene segment has a unique NP binding profile with areas that are enriched for NP association as well as free of NP-binding. Interestingly, NP-vRNA binding profiles have some conservation between influenza A viruses, H1N1 and H3N2, but no correlation was observed between influenza A and B viruses. Our study demonstrates the conserved nature of non-uniform NP binding within influenza viruses. Mapping of the NP-bound vRNA segments provides information on the flexible NP regions that may be involved in facilitating assembly.