RESUMO
Circulating tumor DNA (ctDNA) holds promise in resectable esophageal adenocarcinoma (EAC) to predict patient outcome but is not yet sensitive enough to be clinically applicable. Our aim was to combine ctDNA mutation data with shallow whole-genome sequencing (sWGS)-derived copy number tumor fraction estimates (ichorCNA) to improve pathological response and survival prediction in EAC. In total, 111 stage II/III EAC patients with baseline (n = 111), post-neoadjuvant chemoradiotherapy (nCRT) (n = 68), and pre-surgery (n = 92) plasma samples were used for ctDNA characterization. sWGS (<5× coverage) was performed on all time-point samples, and copy number aberrations were estimated using ichorCNA. Baseline and pre-surgery samples were sequenced using a custom amplicon panel for mutation detection. Detection of baseline ctDNA was successful in 44.3% of patients by amplicon sequencing and 10.5% by ichorCNA. Combining both, ctDNA could be detected in 50.5% of patients. Baseline ctDNA positivity was related to higher T stage (cT3, 4) (p = 0.017). There was no relationship between pathological response and baseline ctDNA positivity. However, baseline ctDNA metrics (variant allele frequency > 1% or ichorCNA > 3%) were associated with a high risk of disease progression [HR = 2.23 (95% CI 1.22-4.07), p = 0.007]. The non-clearance of a baseline variant or ichorCNA > 3% in pre-surgery samples was related to early progression [HR = 4.58 (95% CI 2.22-9.46), p < 0.001]. Multi-signal analysis improves detection of ctDNA and can be used for prognostication of resectable EAC patients. Future studies should explore the potential of multi-modality sequencing for risk stratification and treatment adaptation based on ctDNA results. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
Assuntos
Adenocarcinoma , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias Esofágicas , Humanos , Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/genética , Adenocarcinoma/genética , Adenocarcinoma/terapia , Adenocarcinoma/diagnóstico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/terapia , Biomarcadores Tumorais/genética , MutaçãoRESUMO
BACKGROUND: Assays that account for the biological properties and fragmentation of cell-free DNA (cfDNA) can improve the performance of liquid biopsy. However, preanalytic and physiological differences between individuals on fragmentomic analysis are poorly defined. METHODS: We analyzed the impact of collection tube, plasma processing time, and physiology on the size distribution of cfDNA, their genome-wide representation, and sequence diversity at the cfDNA fragment ends using shallow whole-genome sequencing. RESULTS: Neither different stabilizing collection tubes nor processing times affected the cfDNA fragment sizes, but could impact the genome-wide fragmentation patterns and fragment-end sequences of cfDNA. In addition, beyond differences depending on the gender, the physiological conditions tested between 63 individuals (age, body mass index, use of medication, and chronic conditions) minimally influenced the outcome of fragmentomic methods. CONCLUSIONS: Fragmentomic approaches have potential for implementation in the clinic, pending clear traceability of analytical and physiological factors.
Assuntos
Ácidos Nucleicos Livres , Ácidos Nucleicos Livres/genética , Fragmentação do DNA , Humanos , Biópsia Líquida/métodosRESUMO
BACKGROUND: Epstein-Barr virus (EBV) is an important human pathogenic gammaherpesvirus with carcinogenic potential. The EBV transcriptome has previously been analyzed using both Illumina-based short read-sequencing and Pacific Biosciences RS II-based long-read sequencing technologies. Since the various sequencing methods have distinct strengths and limitations, the use of multiplatform approaches have proven to be valuable. The aim of this study is to provide a more complete picture on the transcriptomic architecture of EBV. METHODS: In this work, we apply the Oxford Nanopore Technologies MinION (long-read sequencing) platform for the generation of novel transcriptomic data, and integrate these with other's data generated by another LRS approach, Pacific BioSciences RSII sequencing and Illumina CAGE-Seq and Poly(A)-Seq approaches. Both amplified and non-amplified cDNA sequencings were applied for the generation of sequencing reads, including both oligo-d(T) and random oligonucleotide-primed reverse transcription. EBV transcripts are identified and annotated using the LoRTIA software suite developed in our laboratory. RESULTS: This study detected novel genes embedded into longer host genes containing 5'-truncated in-frame open reading frames, which potentially encode N-terminally truncated proteins. We also detected a number of novel non-coding RNAs and transcript length isoforms encoded by the same genes but differing in their start and/or end sites. This study also reports the discovery of novel splice isoforms, many of which may represent altered coding potential, and of novel replication-origin-associated transcripts. Additionally, novel mono- and multigenic transcripts were identified. An intricate meshwork of transcriptional overlaps was revealed. CONCLUSIONS: An integrative approach applying multi-technique sequencing technologies is suitable for reliable identification of complex transcriptomes because each techniques has different advantages and limitations, and the they can be used for the validation of the results obtained by a particular approach.
Assuntos
Infecções por Vírus Epstein-Barr , Transcriptoma , Infecções por Vírus Epstein-Barr/genética , Perfilação da Expressão Gênica , Herpesvirus Humano 4/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Fases de Leitura AbertaRESUMO
BACKGROUND: Alternative polyadenylation is commonly examined using cDNA sequencing, which is known to be affected by template-switching artifacts. However, the effects of such template-switching artifacts on alternative polyadenylation are generally disregarded, while alternative polyadenylation artifacts are attributed to internal priming. RESULTS: Here, we analyzed both long-read cDNA sequencing and direct RNA sequencing data of two organisms, generated by different sequencing platforms. We developed a filtering algorithm which takes into consideration that template-switching can be a source of artifactual polyadenylation when filtering out spurious polyadenylation sites. The algorithm outperformed the conventional internal priming filters based on comparison to direct RNA sequencing data. We also showed that the polyadenylation artifacts arise in cDNA sequencing at consecutive stretches of as few as three adenines. There was no substantial difference between the lengths of poly(A) tails at the artifactual and the true transcriptional end sites even though it is expected that internal priming artifacts have shorter poly(A) tails than genuine polyadenylated reads. CONCLUSIONS: Our findings suggest that template switching plays an important role in the generation of spurious polyadenylation and support the need for more rigorous filtering of artifactual polyadenylation sites in cDNA data, or that alternative polyadenylation should be annotated using native RNA sequencing.
Assuntos
Poliadenilação , Artefatos , DNA Complementar/genética , Análise de Sequência de DNA , Transcrição GênicaRESUMO
The role of RNA molecules in the priming of DNA replication and in providing a template for telomerase extension has been known for decades. Since then, several transcripts have been discovered, which play diverse roles in governing replication, including regulation of RNA primer formation, the recruitment of replication origin (Ori) recognition complex, and the assembly of replication fork. Recent studies on viral transcriptomes have revealed novel classes of replication-associated (ra)RNAs, which are expressed from the genomic locations in close vicinity to the Ori. Many of them overlap the Ori, whereas others are terminated close to the replication origin. These novel transcripts can be both protein-coding and non-coding RNAs. The Ori-overlapping part of the mRNAs is generally either the 5'-untranslated regions (UTRs), or the 3'-UTRs of the longer isoforms. Several raRNAs have been identified in various viral families using primarily third-generation long-read sequencing. Hyper-editing of these transcripts has also been described.
Assuntos
Regulação Viral da Expressão Gênica , Transcrição Gênica , Fenômenos Fisiológicos Virais , Replicação Viral/genética , Vírus/genética , Animais , Epistasia Genética , Eucariotos , Redes Reguladoras de Genes , Humanos , Células Procarióticas , Ligação Proteica , Interferência de RNARESUMO
BACKGROUND: Varicella zoster virus (VZV) is a human pathogenic alphaherpesvirus harboring a relatively large DNA molecule. The VZV transcriptome has already been analyzed by microarray and short-read sequencing analyses. However, both approaches have substantial limitations when used for structural characterization of transcript isoforms, even if supplemented with primer extension or other techniques. Among others, they are inefficient in distinguishing between embedded RNA molecules, transcript isoforms, including splice and length variants, as well as between alternative polycistronic transcripts. It has been demonstrated in several studies that long-read sequencing is able to circumvent these problems. RESULTS: In this work, we report the analysis of the VZV lytic transcriptome using the Oxford Nanopore Technologies sequencing platform. These investigations have led to the identification of 114 novel transcripts, including mRNAs, non-coding RNAs, polycistronic RNAs and complex transcripts, as well as 10 novel spliced transcripts and 25 novel transcription start site isoforms and transcription end site isoforms. A novel class of transcripts, the nroRNAs are described in this study. These transcripts are encoded by the genomic region located in close vicinity to the viral replication origin. We also show that the ORF63 exhibits a complex structural variation encompassing the splice sites of VZV latency transcripts. Additionally, we have detected RNA editing in a novel non-coding RNA molecule. CONCLUSIONS: Our investigations disclosed a composite transcriptomic architecture of VZV, including the discovery of novel RNA molecules and transcript isoforms, as well as a complex meshwork of transcriptional read-throughs and overlaps. The results represent a substantial advance in the annotation of the VZV transcriptome and in understanding the molecular biology of the herpesviruses in general.
Assuntos
Herpesvirus Humano 3/genética , Transcriptoma , Linhagem Celular , Humanos , Fases de Leitura Aberta/genética , Isoformas de Proteínas/genética , Edição de RNA , Splicing de RNA , RNA Mensageiro/química , RNA Mensageiro/metabolismo , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Proteínas Virais/genéticaRESUMO
The structure of cell-free DNA (cfDNA) is altered in the blood of patients with cancer. From whole-genome sequencing, we retrieve the cfDNA fragment-end composition using a new software (FrEIA [fragment end integrated analysis]), as well as the cfDNA size and tumor fraction in three independent cohorts (n = 925 cancer from >10 types and 321 control samples). At 95% specificity, we detect 72% cancer samples using at least one cfDNA measure, including 64% early-stage cancer (n = 220). cfDNA detection correlates with a shorter overall (p = 0.0086) and recurrence-free (p = 0.017) survival in patients with resectable esophageal adenocarcinoma. Integrating cfDNA measures with machine learning in an independent test set (n = 396 cancer, 90 controls) achieve a detection accuracy of 82% and area under the receiver operating characteristic curve of 0.96. In conclusion, harnessing the biological features of cfDNA can improve, at no extra cost, the diagnostic performance of liquid biopsies.
Assuntos
Ácidos Nucleicos Livres , Neoplasias , Humanos , Ácidos Nucleicos Livres/genética , Biomarcadores Tumorais/genética , Genômica , Biópsia Líquida , Curva ROCRESUMO
BACKGROUND: High ovarian cancer mortality rates motivate the development of effective and patient-friendly diagnostics. Here, we explored the potential of molecular testing in patient-friendly samples for ovarian cancer detection. METHODS: Home-collected urine, cervicovaginal self-samples, and clinician-taken cervical scrapes were prospectively collected from 54 patients diagnosed with a highly suspicious ovarian mass (benign n = 25, malignant n = 29). All samples were tested for nine methylation markers, using quantitative methylation-specific PCRs that were verified on ovarian tissue samples, and compared to non-paired patient-friendly samples of 110 age-matched healthy controls. Copy number analysis was performed on a subset of urine samples of ovarian cancer patients by shallow whole-genome sequencing. RESULTS: Three methylation markers are significantly elevated in full void urine of ovarian cancer patients as compared to healthy controls (C2CD4D, P = 0.008; CDO1, P = 0.022; MAL, P = 0.008), of which two are also discriminatory in cervical scrapes (C2CD4D, P = 0.001; CDO1, P = 0.004). When comparing benign and malignant ovarian masses, GHSR shows significantly elevated methylation levels in the urine sediment of ovarian cancer patients (P = 0.024). Other methylation markers demonstrate comparably high methylation levels in benign and malignant ovarian masses. Cervicovaginal self-samples show no elevated methylation levels in patients with ovarian masses as compared to healthy controls. Copy number changes are identified in 4 out of 23 urine samples of ovarian cancer patients. CONCLUSIONS: Our study reveals increased methylation levels of ovarian cancer-associated genes and copy number aberrations in the urine of ovarian cancer patients. Our findings support continued research into urine biomarkers for ovarian cancer detection and highlight the importance of including benign ovarian masses in future studies to develop a clinically useful test.
Ovarian cancer is often found late with limited treatment options. Currently, it is difficult to diagnose ovarian cancer correctly and no recommended early detection or screening methods exist. Our aim was to explore the use of DNA-based tests in patient-friendly samples for ovarian cancer detection. Patient-friendly samples are patient materials that can be collected from home without pain or discomfort, such as self-collected vaginal swabs and urine. Using DNA-based tests, we found that urine of women with ovarian cancer contains ovarian cancer-associated signals. Our findings encourage further development of a potential urine test for ovarian cancer detection. This approach could aid early detection and guide women with ovarian masses to appropriate specialist care.
RESUMO
BACKGROUND: Existing methods to detect tumor signal in liquid biopsy have focused on the analysis of nuclear cell-free DNA (cfDNA). However, non-nuclear cfDNA and in particular mitochondrial DNA (mtDNA) has been understudied. We hypothesize that an increase in mtDNA in plasma could reflect the presence of cancer, and that leveraging cell-free mtDNA could enhance cancer detection. RESULTS: We survey 203 healthy and 664 cancer plasma samples from three collection centers covering 12 cancer types with whole genome sequencing to catalogue the plasma mtDNA fraction. The mtDNA fraction is increased in individuals with cholangiocarcinoma, colorectal, liver, pancreatic, or prostate cancer, in comparison to that in healthy individuals. We detect almost no increase of mtDNA fraction in individuals with other cancer types. The mtDNA fraction in plasma correlates with the cfDNA tumor fraction as determined by somatic mutations and/or copy number aberrations. However, the mtDNA fraction is also elevated in a fraction of patients without an apparent increase in tumor-derived cfDNA. A predictive model integrating mtDNA and copy number analysis increases the area under the curve (AUC) from 0.73 when using copy number alterations alone to an AUC of 0.81. CONCLUSIONS: The mtDNA signal retrieved by whole genome sequencing has the potential to boost the detection of cancer when combined with other tumor-derived signals in liquid biopsies.
Assuntos
Ácidos Nucleicos Livres , Neoplasias da Próstata , Masculino , Humanos , Biópsia Líquida , Mitocôndrias/genética , DNA Mitocondrial/genética , Neoplasias da Próstata/genética , Biomarcadores Tumorais/genéticaRESUMO
Cell-free DNA (cfDNA) can be isolated and sequenced from blood and/or urine of cancer patients. Conventional short-read sequencing lacks deployability and speed and can be biased for short cfDNA fragments. Here, we demonstrate that with Oxford Nanopore Technologies (ONT) sequencing we can achieve delivery of genomic and fragmentomic data from liquid biopsies. Copy number aberrations and cfDNA fragmentation patterns can be determined in less than 24 h from sample collection. The tumor-derived cfDNA fraction calculated from plasma of lung cancer patients and urine of bladder cancer patients was highly correlated (R = 0.98) with the tumor fraction calculated from short-read sequencing of the same samples. cfDNA size profile, fragmentation patterns, fragment-end composition, and nucleosome profiling near transcription start sites in plasma and urine exhibited the typical cfDNA features. Additionally, a high proportion of long tumor-derived cfDNA fragments (> 300 bp) are recovered in plasma and urine using ONT sequencing. ONT sequencing is a cost-effective, fast, and deployable approach for obtaining genomic and fragmentomic results from liquid biopsies, allowing the analysis of previously understudied cfDNA populations.
Assuntos
Ácidos Nucleicos Livres , Neoplasias Pulmonares , Sequenciamento por Nanoporos , Humanos , Ácidos Nucleicos Livres/genética , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Genômica/métodos , Análise de Sequência de DNA , DNA/genética , Biomarcadores Tumorais/genéticaRESUMO
In this study, two long-read sequencing (LRS) techniques, MinION from Oxford Nanopore Technologies and Sequel from the Pacific Biosciences, were used for the transcriptional characterization of a prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus. LRS is able to read full-length RNA molecules, and thereby distinguish between transcript isoforms, mono- and polycistronic RNAs, and overlapping transcripts. Altogether, we detected 875 transcript species, of which 759 were novel and 116 were annotated previously. These RNA molecules include 41 novel putative protein coding transcripts [each containing 5'-truncated in-frame open reading frames (ORFs), 14 monocistronic transcripts, 99 polygenic RNAs, 101 non-coding RNAs, and 504 untranslated region isoforms. This work also identified novel replication origin-associated transcripts, upstream ORFs, cis-regulatory sequences and poly(A) sites. We also detected RNA methylation in 99 viral genes and RNA hyper-editing in the longer 5'-UTR transcript isoform of the canonical ORF 19 transcript.
Assuntos
Baculoviridae/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Isoformas de Proteínas/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genética , Metilação , Nucleopoliedrovírus/genética , Fases de Leitura Aberta , RNA Viral , TATA Box , Regiões não TraduzidasRESUMO
African swine fever virus (ASFV) is a large DNA virus belonging to the Asfarviridae family. Despite its agricultural importance, little is known about the fundamental molecular mechanisms of this pathogen. Short-read sequencing (SRS) can produce a huge amount of high-precision sequencing reads for transcriptomic profiling, but it is inefficient for comprehensively annotating transcriptomes. Long-read sequencing (LRS) can overcome some of SRS's limitations, but it also has drawbacks, such as low-coverage and high error rate. The limitations of the two approaches can be surmounted by the combined use of these techniques. In this study, we used Illumina SRS and Oxford Nanopore Technologies LRS platforms with multiple library preparation methods (amplified and direct cDNA sequencings and native RNA sequencing) for constructing the ASFV transcriptomic atlas. This work identified many novel transcripts and transcript isoforms and annotated the precise termini of previously described RNAs. This study identified a novel species of ASFV transcripts, the replication origin-associated RNAs. Additionally, we discovered several nested genes embedded into larger canonical genes. In contrast to the current view that the ASFV transcripts are monocistronic, we detected a significant extent of polycistronism. A multifaceted meshwork of transcriptional overlaps was also discovered.
Assuntos
Vírus da Febre Suína Africana/genética , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Animais , Células Cultivadas , Biblioteca Gênica , Genoma Viral , Macrófagos Alveolares/virologia , RNA Viral/genética , SuínosRESUMO
Viral transcriptomes that are determined using first- and second-generation sequencing techniques are incomplete. Due to the short read length, these methods are inefficient or fail to distinguish between transcript isoforms, polycistronic RNAs, and transcriptional overlaps and readthroughs. Additionally, these approaches are insensitive for the identification of splice and transcriptional start sites (TSSs) and, in most cases, transcriptional end sites (TESs), especially in transcript isoforms with varying transcript ends, and in multi-spliced transcripts. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. Although vaccinia virus (VACV) does not produce spliced RNAs, its transcriptome has a high diversity of TSSs and TESs, and a high degree of polycistronism that leads to enormous complexity. We applied single-molecule, real-time, and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of VACV gene expression.
RESUMO
Long-read sequencing (LRS), a powerful novel approach, is able to read full-length transcripts and confers a major advantage over the earlier gold standard short-read sequencing in the efficiency of identifying for example polycistronic transcripts and transcript isoforms, including transcript length- and splice variants. In this work, we profile the human cytomegalovirus transcriptome using two third-generation LRS platforms: the Sequel from Pacific BioSciences, and MinION from Oxford Nanopore Technologies. We carried out both cDNA and direct RNA sequencing, and applied the LoRTIA software, developed in our laboratory, for the transcript annotations. This study identified a large number of novel transcript variants, including splice isoforms and transcript start and end site isoforms, as well as putative mRNAs with truncated in-frame ORFs (located within the larger ORFs of the canonical mRNAs), which potentially encode N-terminally truncated polypeptides. Our work also disclosed a highly complex meshwork of transcriptional read-throughs and overlaps.
Assuntos
Citomegalovirus/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Citomegalovirus/isolamento & purificação , DNA Complementar , Genes Virais , Humanos , Fases de Leitura Aberta , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , SoftwareRESUMO
OBJECTIVE: In this study, we applied two long-read sequencing (LRS) approaches, including single-molecule real-time and nanopore-based sequencing methods to investigate the time-lapse transcriptome patterns of host gene expression as a response to Vaccinia virus infection. Transcriptomes determined using short-read sequencing approaches are incomplete because these platforms are inefficient or fail to distinguish between polycistronic RNAs, transcript isoforms, transcriptional start sites, as well as transcriptional readthroughs and overlaps. Long-read sequencing is able to read full-length nucleic acids and can therefore be used to assemble complete transcriptome atlases. RESULTS: In this work, we identified a number of novel transcripts and transcript isoforms of Chlorocebus sabaeus. Additionally, analysis of the most abundant 768 host transcripts revealed a significant overrepresentation of the class of genes in the "regulation of signaling receptor activity" Gene Ontology annotation as a result of viral infection.
Assuntos
Perfilação da Expressão Gênica , Infecções por Poxviridae , Animais , Chlorocebus aethiops , Sequenciamento de Nucleotídeos em Larga Escala , Anotação de Sequência Molecular , Isoformas de Proteínas/genética , TranscriptomaRESUMO
Third-generation sequencing is able to read full-length transcripts and thus to efficiently identify RNA molecules and transcript isoforms, including transcript length and splice isoforms. In this study, we report the time-course profiling of the effect of bovine alphaherpesvirus type 1 on the gene expression of bovine epithelial cells using direct cDNA sequencing carried out on MinION device of Oxford Nanopore Technologies. These investigations revealed a substantial up- and down-regulatory effect of the virus on several gene networks of the host cells, including those that are associated with antiviral response, as well as with viral transcription and translation. Additionally, we report a large number of novel bovine transcript isoforms identified by nanopore and synthetic long-read sequencing. This study demonstrates that viral infection causes differential expression of host transcript isoforms. We could not detect an increased rate of transcriptional readthroughs as described in another alphaherpesvirus. According to our knowledge, this is the first report on the use of LoopSeq for the analysis of eukaryotic transcriptomes. This is also the first report on the application of nanopore sequencing for the kinetic characterization of cellular transcriptomes. This study also demonstrates the utility of nanopore sequencing for the characterization of dynamic transcriptomes in any organisms.
Assuntos
Nanoporos , Transcriptoma/genética , Perfilação da Expressão Gênica/métodos , Isoformas de Proteínas/genética , Análise de Sequência de RNA/métodosRESUMO
In this meta-analysis, we re-analysed and compared herpes simplex virus type 1 transcriptomic data generated by eight studies using various short- and long-read sequencing techniques and different library preparation methods. We identified a large number of novel mRNAs, non-coding RNAs and transcript isoforms, and validated many previously published transcripts. Here, we present the most complete HSV-1 transcriptome to date. Furthermore, we also demonstrate that various sequencing techniques, including both cDNA and direct RNA sequencing approaches, are error-prone, which can be circumvented by using integrated approaches. This work draws attention to the need for using multiple sequencing approaches and meta-analyses in transcriptome profiling studies to obtain reliable results.
Assuntos
Perfilação da Expressão Gênica/métodos , Herpesvirus Humano 1/genética , Transcriptoma , Conjuntos de Dados como Assunto , Biblioteca Gênica , Análise de Sequência de RNARESUMO
Characterization of global transcriptomes using conventional short-read sequencing is challenging due to the insensitivity of these platforms to transcripts isoforms, multigenic RNA molecules, and transcriptional overlaps. Long-read sequencing (LRS) can overcome these limitations by reading full-length transcripts. Employment of these technologies has led to the redefinition of transcriptional complexities in reported organisms. In this study, we applied LRS platforms from Pacific Biosciences and Oxford Nanopore Technologies to profile the vaccinia virus (VACV) transcriptome. We performed cDNA and direct RNA sequencing analyses and revealed an extremely complex transcriptional landscape of this virus. In particular, VACV genes produce large numbers of transcript isoforms that vary in their start and termination sites. A significant fraction of VACV transcripts start or end within coding regions of neighbouring genes. This study provides new insights into the transcriptomic profile of this viral pathogen.
Assuntos
Transcriptoma/genética , Vaccinia virus/genética , Vaccinia virus/patogenicidade , Genes Virais/genética , Transcrição Gênica/genéticaRESUMO
Long-read sequencing (LRS) has become a standard approach for transcriptome analysis in recent years. Bovine alphaherpesvirus 1 (BoHV-1) is an important pathogen of cattle worldwide. This study reports the profiling of the dynamic lytic transcriptome of BoHV-1 using two long-read sequencing (LRS) techniques, the Oxford Nanopore Technologies MinION, and the LoopSeq synthetic LRS methods, using multiple library preparation protocols. In this work, we annotated viral mRNAs and non-coding transcripts, and a large number of transcript isoforms, including transcription start and end sites, as well as splice variants of BoHV-1. Our analysis demonstrated an extremely complex pattern of transcriptional overlaps.
Assuntos
Perfilação da Expressão Gênica , Herpesvirus Bovino 1/genética , Sequenciamento de Nucleotídeos em Larga Escala , Transcriptoma/genética , Processamento Alternativo/genética , Sequência de Bases , Linhagem Celular , Regulação Viral da Expressão Gênica , Genoma Viral , Íntrons/genética , Cinética , Anotação de Sequência Molecular , Peptídeos/metabolismo , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Long-read sequencing (LRS) has become increasingly popular due to its strengths in de novo assembly and in resolving complex DNA regions as well as in determining full-length RNA molecules. Two important LRS technologies have been developed during the past few years, including single-molecule, real-time sequencing by Pacific Biosciences, and nanopore sequencing by Oxford Nanopore Technologies. Although current LRS methods produce lower coverage, and are more error prone than short-read sequencing, these methods continue to be superior in identifying transcript isoforms including multispliced RNAs and transcript-length variants as well as overlapping transcripts and alternative polycistronic RNA molecules. Viruses have small, compact genomes and therefore these organisms are ideal subjects for transcriptome analysis with the relatively low-throughput LRS techniques. Recent LRS studies have multiplied the number of previously known transcripts and have revealed complex networks of transcriptional overlaps in the examined viruses.