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Through immune memory, infections have a lasting effect on the host. While memory cells enable accelerated and enhanced responses upon rechallenge with the same pathogen, their impact on susceptibility to unrelated diseases is unclear. We identify a subset of memory T helper 1 (Th1) cells termed innate acting memory T (TIA) cells that originate from a viral infection and produce IFN-γ with innate kinetics upon heterologous challenge in vivo. Activation of memory TIA cells is induced in response to IL-12 in combination with IL-18 or IL-33 but is TCR independent. Rapid IFN-γ production by memory TIA cells is protective in subsequent heterologous challenge with the bacterial pathogen Legionella pneumophila. In contrast, antigen-independent reactivation of CD4+ memory TIA cells accelerates disease onset in an autoimmune model of multiple sclerosis. Our findings demonstrate that memory Th1 cells can acquire additional TCR-independent functionality to mount rapid, innate-like responses that modulate susceptibility to heterologous challenges.
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Imunidade Inata , Memória Imunológica , Interferon gama , Células Th1 , Células Th1/imunologia , Animais , Memória Imunológica/imunologia , Camundongos , Interferon gama/metabolismo , Interferon gama/imunologia , Células T de Memória/imunologia , Camundongos Endogâmicos C57BL , Legionella pneumophila/imunologia , Esclerose Múltipla/imunologia , Interleucina-12/metabolismo , Interleucina-12/imunologiaRESUMO
SUMMARY: Method development for the analysis of cell-free DNA (cfDNA) sequencing data is impeded by limited data sharing due to the strict control of sensitive genomic data. An existing solution for facilitating data sharing removes nucleotide-level information from raw cfDNA sequencing data, keeping alignment coordinates only. This simplified format can be publicly shared and would, theoretically, suffice for common functional analyses of cfDNA data. However, current bioinformatics software requires nucleotide-level information and cannot process the simplified format. We present Fragmentstein, a command-line tool for converting non-sensitive cfDNA-fragmentation data into alignment mapping (BAM) files. Fragmentstein complements fragment coordinates with sequence information from a reference genome to reconstruct BAM files. We demonstrate the utility of Fragmentstein by showing the feasibility of copy number variant (CNV), nucleosome occupancy, and fragment length analyses from non-sensitive fragmentation data. AVAILABILITY AND IMPLEMENTATION: Implemented in bash, Fragmentstein is available at https://github.com/uzh-dqbm-cmi/fragmentstein, licensed under GNU GPLv3.
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Ácidos Nucleicos Livres , Software , Genômica , Genoma , Nucleotídeos , Análise de Sequência de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
BACKGROUND: Basal cell carcinoma (BCC) is the most frequently diagnosed skin cancer and the most common malignancy in humans. Different morphological subtypes of BCC are associated with low- or high-risk of recurrence and aggressiveness, but the underlying biology of how the individual subtypes arise remains largely unknown. Because the majority of BCCs appear to arise from mutations in the same pathway, we hypothesized that BCC development, growth and invasive potential is also influenced by the tumor microenvironment and in particular by cancer-associated fibroblasts (CAFs) and their secreted factors. OBJECTIVE: We aimed to characterize the stroma of the different BCC subtypes with a focus on CAF populations. METHODS: To investigate the stromal features of the different BCC subtypes, we applied laser-capture microdissection (LCM) followed by RNA sequencing. A cohort of 15 BCC samples from 5 different "pure" subtypes (superficial, nodular, micronodular, sclerosing and basosquamous; n=3 each) were selected and included in the analysis. Healthy skin was used as a control (n=6). We confirmed the results by immunohistochemistry. We validated our findings in two independent, public single-cell RNA sequencing (scRNAseq) datasets and by RNAscope. RESULTS: The stroma of the different BCC subtypes have distinct gene expression signatures. Nodular and micronodular seem to have the most similar signatures, while superficial and sclerosing the most different. By comparing low- and high-risk BCC subtypes, we observed that Collagen 10A1 (COL10A1) is overexpressed in the stroma of sclerosing/infiltrative and basosquamous but not micronodular high-risk subtypes. Those findings were confirmed by immunohistochemistry in a cohort of 89 different BCC and 13 healthy skin samples. Moreover, scRNAseq analysis of BCCs of two independent datasets showed that the COL10A1-expressing population of cells is associated with the stroma adjacent to invasive BCC and shows extracellular matrix remodeling features. CONCLUSION: We identified COL10A1 as a marker of high-risk BCC, in particular of the sclerosing/infiltrative and basosquamous subtypes. We demonstrated at the single cell level that COL10A1 is expressed by a specific CAF population associated with the stroma of invasive BCC. This opens up new tailored treatment options as well as a new prognostic biomarker for BCC progression.
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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.
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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.
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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
The temporal coordination of viral gene expression is imperative for the regulation of the herpesvirus replication cycle. While the main factors of this transcriptional coordination are known, the subtler control mechanisms of gene expression remain elusive. Recent long read sequencing-based approached have revealed an intricate meshwork of overlaps between the herpesvirus transcripts and the overlap of the replication origins with noncoding RNAs. It has been shown that the transcriptional apparatuses can physically interfere with one another while transcribing overlapping regions. We hypothesize that transcriptional interference regulates the global gene expression across the herpesvirus genome. Additionally, an overall decrease in transcriptional activity in individual viral genes has been observed following the onset of DNA replication. An overlap of the replication origins with specific transcripts has also been described in several herpesviruses. The genome-wide interactions between the transcriptional apparatuses and between the replication and transcriptional machineries suggest the existence of novel layers of genetic regulation.
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DNA Viral/biossíntese , Herpesviridae/genética , RNA Viral/biossíntese , Origem de Replicação/genética , Replicação do DNA/genética , DNA Viral/genética , Regulação Viral da Expressão Gênica , Redes Reguladoras de Genes/genética , Genoma Viral/genética , RNA Viral/genéticaRESUMO
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.
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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
BACKGROUND AND PURPOSE: Current radiotherapy guidelines rely heavily on imaging-based monitoring. Liquid biopsy monitoring promises to complement imaging by providing frequent systemic information about the tumor. In particular, cell-free DNA (cfDNA) sequencing offers a tumor-agnostic approach, which lends itself to monitoring heterogeneous cohorts of cancer patients. METHODS: We collected plasma cfDNA from oligometastatic patients (OMD) and head-and-neck cancer patients (SCCHN) at six time points before, during, and after radiotherapy, and compared them to the plasma samples of healthy and polymetastatic volunteers. We performed low-pass (on average 7x) whole-genome sequencing on 93 plasma cfDNA samples and correlated copy number alterations and fragment length distributions to clinical and imaging findings. RESULTS: We observed copy number alterations in 4/7 polymetastatic cancer patients, 1/7 OMD and 1/7 SCCHN patients, these patients' imaging showed progression following radiotherapy. Using unsupervised learning, we identified cancer-specific fragment length features that showed a strong correlation with copy number-based tumor fraction estimates. In 4/4 HPV-positive SCCHN patient samples, we detected viral DNA that enabled the monitoring of very low tumor fraction samples. CONCLUSIONS: Our results indicate that an elevated tumor fraction is associated with tumor aggressiveness and systemic tumor spread. This information may be used to adapt treatment strategies. Further, we show that by detecting specific sequences such as viral DNA, the sensitivity of detecting cancer from cell-free DNA sequencing data can be greatly increased.
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Ácidos Nucleicos Livres , Neoplasias de Cabeça e Pescoço , Sequenciamento Completo do Genoma , Humanos , Neoplasias de Cabeça e Pescoço/radioterapia , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/sangue , Ácidos Nucleicos Livres/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Variações do Número de Cópias de DNA , Dosagem Radioterapêutica , Adulto , Carcinoma de Células Escamosas de Cabeça e Pescoço/radioterapia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/sangueRESUMO
Ultra-precision turning is a crucial process in the manufacturing industry as it helps to produce parts with high dimensional accuracy, surface finish, and tolerance. The process is similar to traditional turning but is carried out under special circumstances to achieve greater precision and surface finish. The process can be applied to conventional structural materials, but the demand for machining hardened steels is increasing. The optimization of ultra-precision turning of AISI D2 using cubic boron nitride (CBN) tools is a crucial aspect in the field of high-quality machining. This study aims to evaluate the performance of the process and identify the optimal parameters that result in the best quality components while using a CBN tool's ultra-precision turning of AISI D2. Ultra-precision turning process factors such as cutting speed, feed, and depth of cut were experimentally investigated to enhance the response output, such as surface roughness and cutting force components. The full factorial experimental design was used for determining the process characteristics under different conditions, and experimental results were applied to search for the optimum response of machining performance. The optimization process was done by combining the hybrid genetic algorithm-response surface methodology (GA-RSM) and the Taguchi-grey relational analysis (GRA) statistical tools. These methods are useful in situations where the relationship between the input variables and the output responses is complex and non-linear. The results showed that a hybrid GA-RSM approach, combined with Taguchi-GRA statistical analysis, can effectively find optimal process parameters, leading to the best combination of surface roughness and cutting force. In hybrid Taguchi - GRA, the optimal cutting conditions were found to be a cutting speed of 175 m/min, a feed of 0.025 mm, and a depth of cut of 0.06 mm. The findings of this study provide valuable insights for the optimization of ultra-precision CBN turning operations, contribute to the development of precision manufacturing technology, and can be used as a reference for similar machining processes.
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The success of prime editing depends on the prime editing guide RNA (pegRNA) design and target locus. Here, we developed machine learning models that reliably predict prime editing efficiency. PRIDICT2.0 assesses the performance of pegRNAs for all edit types up to 15 bp in length in mismatch repair-deficient and mismatch repair-proficient cell lines and in vivo in primary cells. With ePRIDICT, we further developed a model that quantifies how local chromatin environments impact prime editing rates.
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Prime editing is a versatile genome editing tool but requires experimental optimization of the prime editing guide RNA (pegRNA) to achieve high editing efficiency. Here we conducted a high-throughput screen to analyze prime editing outcomes of 92,423 pegRNAs on a highly diverse set of 13,349 human pathogenic mutations that include base substitutions, insertions and deletions. Based on this dataset, we identified sequence context features that influence prime editing and trained PRIDICT (prime editing guide prediction), an attention-based bidirectional recurrent neural network. PRIDICT reliably predicts editing rates for all small-sized genetic changes with a Spearman's R of 0.85 and 0.78 for intended and unintended edits, respectively. We validated PRIDICT on endogenous editing sites as well as an external dataset and showed that pegRNAs with high (>70) versus low (<70) PRIDICT scores showed substantially increased prime editing efficiencies in different cell types in vitro (12-fold) and in hepatocytes in vivo (tenfold), highlighting the value of PRIDICT for basic and for translational research applications.
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Aprendizado Profundo , Humanos , Edição de Genes , Hepatócitos , Mutação , Redes Neurais de Computação , Sistemas CRISPR-Cas/genéticaRESUMO
Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. SIGNIFICANCE: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.
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Melanoma , Neoplasias Cutâneas , Humanos , Espécies Reativas de Oxigênio , Proteínas Proto-Oncogênicas B-raf/genética , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Linhagem Celular Tumoral , Mutação , Proteínas de Membrana/genética , GTP Fosfo-Hidrolases/genéticaRESUMO
Biobanking of surplus human healthy and disease-derived tissues is essential for diagnostics and translational research. An enormous amount of formalin-fixed and paraffin-embedded (FFPE), Tissue-Tek OCT embedded or snap-frozen tissues are preserved in many biobanks worldwide and have been the basis of translational studies. However, their usage is limited to assays that do not require viable cells. The access to intact and viable human material is a prerequisite for translational validation of basic research, for novel therapeutic target discovery, and functional testing. Here we show that surplus tissues from multiple solid human cancers directly slow-frozen after resection can subsequently be used for different types of methods including the establishment of 2D, 3D, and ex vivo cultures as well as single-cell RNA sequencing with similar results when compared to freshly analyzed material.
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Formaldeído , Neoplasias , Humanos , Inclusão em Parafina , Bancos de Espécimes Biológicos , Sequenciamento do ExomaRESUMO
PDE4B (phosphodiesterase-4B) has an important role in cancer and in pharmacology of some disorders, such as inflammatory diseases. Remarkably in Native Americans, PDE4B variants are associated with acute lymphoblastic leukemia (ALL) relapse, as this gene modulates sensitivity of glucocorticoids used in ALL chemotherapy. PDE4B allele rs6683977.G, associated with genomic regions of Native American origin in US-Hispanics (admixed among Native Americans, Europeans, and Africans), increases ALL relapse risk, contributing to an association between Native American ancestry and ALL relapse that disappeared with an extra-phase of chemotherapy. This result insinuates that indigenous populations along the Americas may have high frequencies of rs6683977.G, but this has never been corroborated. We studied ancestry and PDE4B diversity in 951 healthy individuals from nine Latin American populations. In non-admixed Native American populations rs6683977.G has frequencies greater than 90%, is in linkage disequilibrium with other ALL relapse associated and regulatory variants in PDE4B-intron-7, conforming haplotypes showing their highest worldwide frequencies in Native Americans (>0.82). Our findings inform the discussion on the pertinence of an extra-phase of chemotherapy in Native American populations, and exemplifies how knowledge generated in US-Hispanics is relevant for their even more neglected and vulnerable Native American ancestors along the American continent.
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Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Neoplasias , Farmacogenética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Genética Populacional , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Recidiva , Indígena Americano ou Nativo do AlascaRESUMO
High cell viability and recovered cell concentration are typical quality control requirements for single-cell processing and quality data. This protocol describes procedures for sampling, live-cell biobanking, preprocessing for single-cell RNA sequencing, and analysis of fine-needle aspiration (FNA) samples of the skin. The minimally invasive nature of FNA collection is more accepted by patients and allows for frequent longitudinal sampling, resulting in high-quality single-cell sequencing data that capture cellular heterogeneity in clinical samples.
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Biópsia por Agulha Fina/métodos , Análise de Dados , Análise de Célula Única/métodos , Manejo de Espécimes/métodos , Humanos , Análise de Sequência de RNA/métodosRESUMO
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.
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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.
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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
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.
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Talimogene laherparepvec (T-VEC) is a genetically modified herpes simplex 1 virus (HSV-1) approved for cancer therapy. We investigate its effect on the clinical, histological, single-cell transcriptomic, and immune repertoire level using repeated fine-needle aspirates (FNAs) of injected and noninjected lesions in primary cutaneous B cell lymphoma (pCBCL). Thirteen patients received intralesional T-VEC, 11 of which demonstrate tumor response in the injected lesions. Using single-cell sequencing of the FNAs, we identify the malignant population and separate three pCBCL subtypes. Twenty-four hours after the injection, we detect HSV-1T-VEC transcripts in malignant and nonmalignant cells of the injected lesion but not of the noninjected lesion. Oncolytic virotherapy results in a rapid eradication of malignant cells. It also leads to interferon pathway activation and early influx of natural killer cells, monocytes, and dendritic cells. These events are followed by enrichment in cytotoxic T cells and a decrease of regulatory T cells in injected and noninjected lesions.
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Linfoma de Células B/imunologia , Linfoma de Células B/terapia , Vírus Oncolíticos/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Produtos Biológicos/imunologia , Células Dendríticas/imunologia , Feminino , Herpesvirus Humano 1/imunologia , Humanos , Células Matadoras Naturais/imunologia , Linfoma de Células B/virologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Terapia Viral Oncolítica/métodos , Análise de Célula Única , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/imunologiaRESUMO
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.