Comprehensive characterization of differentially spliced RNA transcripts with nanopore sequencing is limited by bioinformatics tools that are reliant on existing annotations. We have developed FLAME, a bioinformatics pipeline for alternative splicing analysis of gene-specific or transcriptome-wide longread sequencing data. FLAME is a Python-based tool aimed at providing comprehensible quantification of full-length splice variants, reliable de novo recognition of splice sites and exons, and representation of consecutive exon connectivity in the form of a weighted adjacency matrix. Notably, this workflow circumvents issues related to inadequate reference annotations and allows for incorporation of shortread sequencing data to improve the confidence of nanopore sequencing reads. In this study, the Epstein-Barr virus long non-coding RNA RPMS1 was used to demonstrate the utility of the pipeline. RPMS1 is ubiquitously expressed in Epstein-Barr virus associated cancer and known to undergo ample differential splicing. To fully resolve the RPMS1 spliceome, we combined gene-specific nanopore sequencing reads from a primary gastric adenocarcinoma and a nasopharyngeal carcinoma cell line with matched publicly available short-read sequencing datasets. All previously reported splice variants, including putative ORFs, were detected using FLAME. In addition, 32 novel exons, including two intron retentions and a cassette exon, were discovered within the RPMS1 gene.
Epstein-Barr virus (EBV) DNA is regularly found in the blood of patients with EBV associated diseases and occasionally in healthy individuals. However, EBV infected primary B-lymphocytes have not yet been detected using scRNA seq. Here, we screened the viral transcriptome in single cell RNA sequencing datasets from peripheral blood to identify virus infected cells. Whereas EBV RNA was detected in an immunocompromised patient, EBV associated nasopharyngeal carcinoma and multiple sclerosis samples did not display any levels of circulating EBV RNA. We further screened whole-blood samples from a cohort of immunosuppressed patients for viral transcripts using a custom enhanced RT-qPCR panel and detected latency programs dominated by noncoding RNAs (EBERs and RPMS1). To explore the interplay between the EBV and the host-cell transcriptome profile, we used enriched B-lymphocytes from a splenectomy patient with 30% EBER positivity estimated by in situ hybridization and performed 5-prime single-cell RNA sequencing with paired VDJ profiling. The EBV expression pattern of the patients B-lymphocytes confirmed the RT-qPCR assay with RPMS1 and LMP-1/BNLF2a/b significantly dominating the sequenced EBV polyadenylated RNA. A comparison between the expression profile of EBV positive B-lymphocytes and healthy controls B-lymphocytes revealed the upregulation in genes involved in cell population proliferation when infected with EBV. This is further supported by a measurable polyclonal expansion in the patient, as compared to a control, emphasizing EBVs role in a host-cells tendency for cellular expansion. However, when contrasting to cells that have undergone malignant transformation, the primary EBV infected cells display a rather dissimilar expression profile, even to cells that are supposed to simulate primary EBV infection (I.e. Lymphoblastoid Cell Lines). This implies that during primary infection of EBV, the host-cell enters a state of premalignancy rather than a complete oncogenic transformation at the initial time of infection
Epstein-Barr virus is associated with two types of epithelial neoplasms, nasopharyngeal carcinoma and gastric adenocarcinoma. The viral long non-coding RNA RPMS1 is the most abundantly expressed poly-adenylated viral RNA in these malignant tissues. The RPMS1 gene is known to contain two cassette exons, exon Ia and Ib, and several alternative splicing variants have been described in low-throughput studies. To characterize the entire RPMS1 spliceome we combined long-read sequencing data from the nasopharyngeal cell line C666-1 and a primary gastric adenocarcinoma, with complementary short-read sequencing datasets. We developed FLAME, a Python-based bioinformatics package that can generate complete high resolution characterization of RNA splicing at full-length. Using FLAME, we identified 32 novel exons in the RPMS1 gene, primarily within the large constitutive exons III, V and VII. Two of the novel exons contained retention of the intron between exon III and exon IV, and a novel cassette exon was identified between VI and exon VII. All previously described transcript variants of RPMS1 containing putative ORFs were identified at various levels. Similarly, native transcripts with the potential to form previously reported circular RNA elements were detected. Our work illuminates the multifaceted nature of viral transcriptional repertoires. FLAME provides a comprehensive overview of the relative abundance of alternative splice variants and allows for a wealth of previously unknown splicing events to be unveiled.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.