Splicing Patterns Research Articles

Full-length RNA-Seq of the RHOH gene in human B cells reveals new exons and splicing patterns.

The RhoH protein is a member of the Ras superfamily of guanosine triphosphate-binding proteins. RhoH is an atypical Rho family member that is always GTP-bound and thus always activated. It is restrictively expressed in normal hematopoietic cells, where it is a negative regulator of cell growth and survival. We previously analyzed the RHOH gene structure and demonstrated that this gene is composed of 7 exons, one single encoding exon located at the 3' extremity of the gene, preceded by 6 noncoding exons. To further understand the transcription events associated with this gene, we performed full-length RNA-Seq on 12 B-cell lines. We identified new exons, new splice events and new splice sites, leading to the discovery of 38 RHOH mRNA molecules, 27 of which have never been described before. Here, we also describe new fusion transcripts. Moreover, our method allowed quantitative measurements of the different mRNA species relative to each other in relation to B-cell differentiation.

Abstract B006: Rescuing expression of a tumor suppressor gene – restoring FH in Hereditary leiomyomatosis and renal cell carcinoma

Abstract Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a disorder characterized by the loss of fumarate hydratase (FH) activity, leading to the development of cutaneous leiomyomas, uterine leiomyomas, and aggressive early-onset type 2 papillary renal cell carcinoma. In HLRCC, the disease is driven by the somatic loss of the remaining functional FH allele in individuals carrying a germline pathogenic variant in one copy of the FH gene. The complete loss of FH activity disrupts the tricarboxylic acid (TCA) cycle, resulting in the accumulation of fumarate, which disrupts signaling pathways and contributes to the oncogenic phenotype. We recently identified a single nucleotide polymorphism (SNP) in an intronic region of the fumarate hydratase (FH) gene that leads to the loss of FH activity (Crooks et al., 2023). This SNP introduces a novel splice-acceptor site, resulting in the inclusion of an aberrant exon between wild-type exons 9 and 10. This ‘poison-like’ exon encodes a premature STOP codon, producing a nonfunctional truncated FH protein. Previous studies have shown that restoring FH activity can abrogate pathogenic behavior in patient-derived cells, highlighting the potential for therapeutic interventions targeting FH activity restoration. Our goal is to restore FH enzymatic activity by preventing the inclusion of this cryptic exon in carriers of this variant. To achieve this, we are exploring strategies to restore functional FH production by interfering with the inclusion of the pathogenic intron during splicing by removing the splice acceptor using CRISPR-Cas9 or base editing technologies. We tested these approaches by generating a reporter cell line that recapitulates the splicing pattern observed in patients, where expression of GFP depends on correct splicing between exon 9 and 10. We successfully identified antisense oligonucleotide (ASO) and single-guide RNA (sgRNA) sequences that restore GFP expression in our reporter cell lines. We are now evaluating the efficacy of these reagents in patient-derived cells. An alternative approach involves using synthetic RNA to restore FH expression. The expression of proteins from synthetic RNAs is a promising strategy for treating cancer and infectious diseases. We are currently investigating the efficiency of restoring FH activity through in-vitro transcribed (IVT) mRNA. This approach has the advantage of enabling FH function restoration in cells with mutations that are not amenable to antisense oligonucleotide (ASO) or gene editing techniques. This work serves as an avenue to investigate the application of nucleic acid therapies to restoring activity tumor suppressor genes in kidney cancer. Citation Format: Siddhardha S. Maligireddy, Mariana D. Mandler, Jodie C. Lunger, Christina M. Fitzsimmons, Daniel R. Crooks, Geetha M. Cawthon, Christopher J. Ricketts, Cathy D. Vocke, W. Marson Linehan, Pedro J. Batista. Rescuing expression of a tumor suppressor gene – restoring FH in Hereditary leiomyomatosis and renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr B006.

Targeting splicing for hematological malignancies therapy.

Alterations in splicing patterns of leukemic cells have a functional impact and influence most cellular processes since aberrantly spliced isoforms can provide a proliferative advantage, enable to evade apoptosis, induce metabolic reprogramming, change cell signaling and antitumor immune response, or develop drug resistance. In this Review, we first characterize the general mechanism of mRNA processing regulation with a focus on the role of splicing factors, which are commonly mutated in blood neoplasms. Next, we provide a comprehensive summary on the current understanding of alternative splicing events, which confer resistance to targeted treatment strategies and immunotherapy. We introduce the functional consequences of mis-spliced variants (CD19-∆ex2, CD22-∆ex2, CD22-∆ex5-6, CD33-∆ex2, PIK3CD-S, BCR-ABL35INS, BIM-γ, FPGS-8PR, dCK-∆ex2-3, and SLC29A1-∆ex13) production in leukemic cells. Of therapeutic relevance, we summarize novel strategies focused on pharmacological correction of aberrant splicing, including small-molecule splicing modulators and splice-switching oligonucleotides. We also include the findings of recent preclinical investigation of the antisense strategies based on modified oligonucleotides. Finally, we discuss the potential of emerging combination therapies for the treatment of hematological disorders with disrupted splicing.

RBPWorld for exploring functions and disease associations of RNA-binding proteins across species.

RNA-binding proteins (RBPs) play key roles in a wide range of physiological and pathological processes. To facilitate the investigation of RBP functions and disease associations, we updated the EuRBPDB and renamed it as RBPWorld (http://research.gzsys.org.cn/rbpworld/#/home). Leveraging 998 RNA-binding domains (RBDs) and 87 RNA-binding Proteome (RBPome) datasets, we successfully identified 1 393 413 RBPs from 445 species, including 3030 human RBPs (hRBPs). RBPWorld includes primary RNA targets of diverse hRBPs, as well as potential downstream regulatory pathways and alternative splicing patterns governed by various hRBPs. These insights were derived from analyses of 1515 crosslinking immunoprecipitation-seq datasets and 616 RNA-seq datasets from cells with hRBP gene knockdown or knockout. Furthermore, we systematically identified 929 RBPs with multi-functions, including acting as metabolic enzymes and transcription factors. RBPWorld includes 838 disease-associated hRBPs and 970 hRBPs that interact with 12 disease-causing RNA viruses. This provision allows users to explore the regulatory roles of hRBPs within the context of diseases. Finally, we developed an intuitive interface for RBPWorld, facilitating users easily access all the included data. We believe that RBPWorld will be a valuable resource in advancing our understanding of the biological roles of RBPs across different species.

Phenotypic complexities of rare heterozygous neurexin-1 deletions.

Given the large number of genes significantly associated with risk for neuropsychiatric disorders, a critical unanswered question is the extent to which diverse mutations --sometimes impacting the same gene-- will require tailored therapeutic strategies. Here we consider this in the context of rare neuropsychiatric disorder-associated copy number variants (2p16.3) resulting in heterozygous deletions in NRXN1 , a pre-synaptic cell adhesion protein that serves as a critical synaptic organizer in the brain. Complex patterns of NRXN1 alternative splicing are fundamental to establishing diverse neurocircuitry, vary between the cell types of the brain, and are differentially impacted by unique (non-recurrent) deletions. We contrast the cell-type-specific impact of patient-specific mutations in NRXN1 using human induced pluripotent stem cells, finding that perturbations in NRXN1 splicing result in divergent cell-type-specific synaptic outcomes. Via distinct loss-of-function (LOF) and gain-of-function (GOF) mechanisms, NRXN1 +/- deletions cause decreased synaptic activity in glutamatergic neurons, yet increased synaptic activity in GABAergic neurons. Reciprocal isogenic manipulations causally demonstrate that aberrant splicing drives these changes in synaptic activity. For NRXN1 deletions, and perhaps more broadly, precision medicine will require stratifying patients based on whether their gene mutations act through LOF or GOF mechanisms, in order to achieve individualized restoration of NRXN1 isoform repertoires by increasing wildtype, or ablating mutant isoforms. Given the increasing number of mutations predicted to engender both LOF and GOF mechanisms in brain disorders, our findings add nuance to future considerations of precision medicine.

The splicing factor SF3B1 is essential for proper alternative splicing and zygotic genome activation in early porcine embryos

Alternative splicing (AS) is a pivotal posttranscriptional regulatory mechanism that is involved in embryonic development. However, the roles of AS in specific developmental events, especially the zygotic genome activation (ZGA) of porcine early embryos, remain unclear. In this study, we demonstrated that alternative splicing events (ASEs) were most prevalent in mammalian embryos during ZGA and that skipped exons were the predominant splicing pattern. When splicing factor 3B subunit 1 (SF3B1) was disrupted by the inhibitor pladienolide B (PlaB), we observed that porcine embryos were markedly arrested at the 4-cell stage. Concurrently, the main ZGA genes, namely, DPPA2, EIF6, and SORD, underwent aberrant splicing indicative of the failure of ZGA. Moreover, embryonic metabolic homeostasis was significantly disrupted at the 4-cell stage by SF3B1 inhibition, resulting in increases in the LDHA/LDHB ratio and lactate levels. Interestingly, the levels of the histone lactylation modifications pan-Kla and H4K5la also increased. Our findings enhance our understanding of early mammalian embryonic development, reveal the crucial role of porcine early embryogenesis, and help to resolve reproductive difficulties related to embryonic development.

MDM4 exon skipping upon dysfunctional ribosome assembly

Recent studies revealed how nucleolar stress enhances MDM4 exon skipping and activates p53 via the ribosomal protein L22 (RPL22; eL22). Tumor-associated L22 mutations lead to full-length MDM4 synthesis, overcoming tumor suppression by p53. This forum article explores how MDM4 splicing patterns integrate stress signaling to take p53-dependent cell fate decisions.

Sex-specific mRNA alternative splicing patterns and Dmrt1 isoforms contribute to sex determination and differentiation of oyster

Sex-specific mRNA alternative splicing patterns and Dmrt1 isoforms contribute to sex determination and differentiation of oyster

Unravelling alternative splicing patterns in susceptible and resistant Brassica napus lines in response to Xanthomonas campestris infection

BackgroundRapeseed (Brassica napus L.) is an important oil and industrial crop worldwide. Black rot caused by the bacterial pathogen Xanthomonas campestris pv. campestris (Xcc) is an infectious vascular disease that leads to considerable yield losses in rapeseed. Resistance improvement through genetic breeding is an effective and sustainable approach to control black rot disease in B. napus. However, the molecular mechanisms underlying Brassica-Xcc interactions are not yet fully understood, especially regarding the impact of post-transcriptional gene regulation via alternative splicing (AS).ResultsIn this study, we compared the AS landscapes of a susceptible parental line and two mutagenized B. napus lines with contrasting levels of black rot resistance. Different types of AS events were identified in these B. napus lines at three time points upon Xcc infection, among which intron retention was the most common AS type. A total of 1,932 genes was found to show differential AS patterns between different B. napus lines. Multiple defense-related differential alternative splicing (DAS) hub candidates were pinpointed through an isoform-based co-expression network analysis, including genes involved in pathogen recognition, defense signalling, transcriptional regulation, and oxidation reduction.ConclusionThis study provides new insights into the potential effects of post-transcriptional regulation on immune responses in B. napus towards Xcc attack. These findings could be beneficial for the genetic improvement of B. napus to achieve durable black rot resistance in the future.

Epigenetics and alternative splicing in cancer: old enemies, new perspectives.

In recent years, significant strides in both conceptual understanding and technological capabilities have bolstered our comprehension of the factors underpinning cancer initiation and progression. While substantial insights have unraveled the molecular mechanisms driving carcinogenesis, there has been an overshadowing of the critical contribution made by epigenetic pathways, which works in concert with genetics. Mounting evidence demonstrates cancer as a complex interplay between genetics and epigenetics. Notably, epigenetic elements play a pivotal role in governing alternative pre-mRNA splicing, a primary contributor to protein diversity. In this review, we have provided detailed insights into the bidirectional communication between epigenetic modifiers and alternative splicing, providing examples of specific genes and isoforms affected. Notably, succinct discussion on targeting epigenetic regulators and the potential of the emerging field of epigenome editing to modulate splicing patterns is also presented. In summary, this review offers valuable insights into the intricate interplay between epigenetics and alternative splicing in cancer, paving the way for novel approaches to understanding and targeting this critical process.

Genome-Scale Identification of Wild Soybean Serine/Arginine-Rich Protein Family Genes and Their Responses to Abiotic Stresses.

Serine/arginine-rich (SR) proteins mostly function as splicing factors for pre-mRNA splicing in spliceosomes and play critical roles in plant development and adaptation to environments. However, detailed study about SR proteins in legume plants is still lacking. In this report, we performed a genome-wide investigation of SR protein genes in wild soybean (Glycine soja) and identified a total of 31 GsSR genes from the wild soybean genome. The analyses of chromosome location and synteny show that the GsSRs are unevenly distributed on 15 chromosomes and are mainly under the purifying selection. The GsSR proteins can be phylogenetically classified into six sub-families and are conserved in evolution. Prediction of protein phosphorylation sites indicates that GsSR proteins are highly phosphorylated proteins. The protein-protein interaction network implies that there exist numerous interactions between GsSR proteins. We experimentally confirmed their physical interactions with the representative SR proteins of spliceosome-associated components such as U1-70K or U2AF35 by yeast two-hybrid assays. In addition, we identified various stress-/hormone-responsive cis-acting elements in the promoter regions of these GsSR genes and verified their expression patterns by RT-qPCR analyses. The results show most GsSR genes are highly expressed in root and stem tissues and are responsive to salt and alkali stresses. Splicing analysis showed that the splicing patterns of GsSRs were in a tissue- and stress-dependent manner. Overall, these results will help us to further investigate the biological functions of leguminous plant SR proteins and shed new light on uncovering the regulatory mechanisms of plant SR proteins in growth, development, and stress responses.

An Alternatively Spliced Gain-of-Function NT5C2 Isoform Contributes to Chemoresistance in Acute Lymphoblastic Leukemia.

Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B-ALL patients, suggesting additional mechanisms of resistance. By mining RNA sequencing datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, antifolates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-mercaptopurine in B-ALL cells both in vitro and in vivo. Furthermore, both NT5C2ex6a and the R238W variant induced collateral sensitivity to the inosine monophosphate dehydrogenase inhibitor mizoribine. These results ascribe to splicing perturbations an important role in chemotherapy resistance in relapsed B-ALL and suggest that inosine monophosphate dehydrogenase inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias. Significance: Alternative splicing is a potent mechanism of acquired drug resistance in relapsed/refractory acute lymphoblastic leukemias that has diagnostic and therapeutic implications for patients who lack mutations in known chemoresistance genes.

Mitochondrial Splicing Efficiency Is Lower in Holoparasites Than in Free-Living Plants.

Mitochondria play a crucial role in eukaryotic organisms, housing their own genome with genes vital for oxidative phosphorylation. Coordination between nuclear and mitochondrial genomes is pivotal for organelle gene expression. Splicing, editing and processing of mitochondrial transcripts are regulated by nuclear-encoded factors. Splicing efficiency (SEf) of the many group II introns present in plant mitochondrial genes is critical for mitochondrial function since a splicing defect or splicing deficiency can severely impact plant growth and development. This study investigates SEf in free-living and holoparasitic plants, focusing on 25 group II introns from 15 angiosperm species. Our comparative analyses reveal distinctive splicing patterns with holoparasites exhibiting significantly lower SEf, potentially linked to their unique evolutionary trajectory. Given the preponderance of horizontal gene transfer (HGT) in parasitic plants, we investigated the effect of HGT on SEf, such as the presence of foreign introns or foreign nuclear-encoded splicing factors. Contrary to expectations, the SEf reductions do not correlate with HGT events, suggesting that other factors are at play, such as the loss of photosynthesis or the transition to a holoparasitic lifestyle. The findings of this study broaden our understanding of the molecular evolution in parasitic plants and shed light on the multifaceted factors influencing organelle gene expression.

IDEIS: a tool to identify PTPRC/CD45 isoforms from single-cell transcriptomic data.

Single-cell RNA sequencing (scRNA-seq) methods are widely used in life sciences, including immunology. Typical scRNA-seq analysis pipelines quantify the abundance of particular transcripts without accounting for alternative splicing. However, a well-established pan-leukocyte surface marker, CD45, encoded by the PTPRC gene, presents alternatively spliced variants that define different immune cell subsets. Information about some of the splicing patterns in particular cells in the scRNA-seq data can be obtained using isotype-specific DNA oligo-tagged anti-CD45 antibodies. However, this requires generation of an additional sequencing DNA library. Here, we present IDEIS, an easy-to-use software for CD45 isoform quantification that uses single-cell transcriptomic data as the input. We showed that IDEIS accurately identifies canonical human CD45 isoforms in datasets generated by 10× Genomics 5' sequencing assays. Moreover, we used IDEIS to determine the specificity of the Ptprc splicing pattern in mouse leukocyte subsets.

SRSF6 modulates histone-chaperone HIRA splicing to orchestrate AR and E2F activity in prostate cancer.

Despite novel therapeutic strategies, advanced-stage prostate cancer (PCa) remains highly lethal, pointing out the urgent need for effective therapeutic strategies. While dysregulation of the splicing process is considered a cancer hallmark, the role of certain splicing factors remains unknown in PCa. This study focuses on characterizing the levels and role of SRSF6 in this disease. Comprehensive analyses of SRSF6 alterations (copy number/mRNA/protein) were conducted across eight well-characterized PCa cohorts and the Hi-MYC transgenic model. SRSF6 was up-regulated in PCa samples, correlating with adverse clinical parameters. Functional assays, both in vitro (cell proliferation, migration, colony, and tumorsphere formation) and in vivo (xenograft tumors), demonstrated the impact of SRSF6 modulation on critical cancer hallmarks. Mechanistically, SRSF6 regulates the splicing pattern of the histone-chaperone HIRA, consequently affecting the activity of H3.3 in PCa and breast cancer cell models and disrupting pivotal oncogenic pathways (AR and E2F) in PCa cells. These findings underscore SRSF6 as a promising therapeutic target for PCa/advanced-stage PCa.

Characterization of aberrant splicing in pediatric central nervous system tumors reveals CLK1 as a candidate oncogenic dependency.

Pediatric brain cancer is the leading cause of disease-related mortality in children, and many aggressive tumors still lack effective treatment strategies. We characterized aberrant alternative splicing across pediatric brain tumors, identifying pediatric high-grade gliomas (HGGs) among the most heterogeneous. Annotating these events with UniProt, we identified 11,940 splice events in 5,368 genes leading to potential protein function changes. We discovered CDC-like kinase 1 (CLK1) is aberrantly spliced to include exon 4, resulting in a gain of two phosphorylation sites and subsequent activation. Inhibition of CLK1 with Cirtuvivint significantly decreased both cell viability and proliferation in the pediatric HGG KNS-42 cell line. Morpholino-mediated depletion of CLK1 exon 4 splicing reduced RNA expression, protein abundance, and cell viability with concurrent differential expression of 78 cancer genes and differential splicing at functional sites in 193 cancer genes. Our findings highlight a dependency of pediatric HGGs on CLK1 and represent a promising therapeutic strategy.

Genome-wide identification and gene expression networks of LBD transcription factors in Populus trichocarpa

The Lateral Organ Boundaries Domain (LBD) proteins, an exclusive family of transcription factors (TFs) found solely in plants, play pivotal roles in lateral organogenesis, stress adaptation, secondary growth, and hormonal signaling responses. In this study, a total of 55 PtLBD TFs from Populus trichocarpa were identified and systematically classified into two subfamilies, designated as subfamily-I and subfamily-II with seven distinct groups based on phylogenetic analysis. Gene structure detection indicated that the difference of phase numbers linking adjacent exons contribute to the variations in splicing patterns among different PtLBD groups. Numerous transcription factor binding sites and cis-elements pertinent to hormone signaling pathways and stress response mechanisms were identified within the upstream promoter regions of the PtLBD genes. Thirty-five PtLBDs were found to be engaged in either tandem or segmental duplications, and genomic collinearity analysis revealed a stronger alignment between PtLBD genes and eudicots plants compared to their relationship with monocots. GO enrichment and temporal-spatio expression patterns showed that PtLBD7 from subfamily-I and PtLBD20 from subfamily-II, along with other 13 PtLBDs, were involved in plant growth and development biological processes. The multilayered hierarchical gene networks (ML-hGRN) mediated by PtLBD7 and PtLBD20 indicated that PtLBDs were mainly function in poplar growth and stress tolerance through a multifaceted and intricate regulatory machinery. This study lays a solid groundwork for delving deeper into the roles and underlying mechanisms of LBD transcription factors in poplar, specifically those related to plant hormones and stress tolerance.

An anciently diverged family of RNA binding proteins maintain correct splicing of a class of ultra-long exons through cryptic splice site repression

Previously, we showed that the germ cell-specific nuclear protein RBMXL2 represses cryptic splicing patterns during meiosis and is required for male fertility (Ehrmann et al., 2019). Here, we show that in somatic cells the similar yet ubiquitously expressed RBMX protein has similar functions. RBMX regulates a distinct class of exons that exceed the median human exon size. RBMX protein-RNA interactions are enriched within ultra-long exons, particularly within genes involved in genome stability, and repress the selection of cryptic splice sites that would compromise gene function. The RBMX gene is silenced during male meiosis due to sex chromosome inactivation. To test whether RBMXL2 might replace the function of RBMX during meiosis we induced expression of RBMXL2 and the more distantly related RBMY protein in somatic cells, finding each could rescue aberrant patterns of RNA processing caused by RBMX depletion. The C-terminal disordered domain of RBMXL2 is sufficient to rescue proper splicing control after RBMX depletion. Our data indicate that RBMX and RBMXL2 have parallel roles in somatic tissues and the germline that must have been conserved for at least 200 million years of mammalian evolution. We propose RBMX family proteins are particularly important for the splicing inclusion of some ultra-long exons with increased intrinsic susceptibility to cryptic splice site selection.

PRMT1 Modulates Alternative Splicing to Enhance HPV18 mRNA Stability and Promote the Establishment of Infection.

Only persistent HPV infections lead to the development of cancer. Thus, understanding the virus-host interplay that influences the establishment of viral infection has important implications for HPV biology and human cancers. The ability of papillomaviruses to establish in cells requires the strict temporal regulation of viral gene expression in sync with cellular differentiation. This control primarily happens at the level of RNA splicing and polyadenylation. However, the details of how this spatio-temporal regulation is achieved still need to be fully understood. Until recently, it has been challenging to study the early events of the HPV lifecycle following infection. We used a single-cell genomics approach to identify cellular factors involved in viral infection and establishment. We identify protein arginine N-methyltransferase 1 (PRMT1) as an important factor in viral infection of primary human cervical cells. PRMT1 is the main cellular enzyme responsible for asymmetric dimethylation of cellular proteins. PRMT1 is an enzyme responsible for catalyzing the methylation of arginine residues on various proteins, which influences processes such as RNA processing, transcriptional regulation, and signal transduction. In this study, we show that HPV18 infection leads to increased PRMT1 levels across the viral lifecycle. PRMT1 is critical for the establishment of a persistent infection in primary cells. Mechanistically, PRMT1 inhibition leads to a highly dysregulated viral splicing pattern. Specifically, reduced PRMT1 activity leads to intron retention and a change in the E6 and E7 expression ratio. In the absence of PRMT1, viral transcripts are destabilized and subject to degradation via the nonsense-mediated decay (NMD) pathway. These findings highlight PRMT1 as a critical regulator of the HPV18 lifecycle, particularly in RNA processing, and position it as a potential therapeutic target for persistent HPV18 infections.

SQANTI-reads: a tool for the quality assessment of long read data in multi-sample lrRNA-seq experiments.

SQANTI-reads leverages SQANTI3, a tool for the analysis of the quality of transcript models, to develop a read-level quality control framework for replicated long-read RNA-seq experiments. The number and distribution of reads, as well as the number and distribution of unique junction chains (transcript splicing patterns), in SQANTI3 structural categories are informative of raw data quality. Multi-sample visualizations of QC metrics are presented by experimental design factors to identify outliers. We introduce new metrics for 1) the identification of potentially under-annotated genes and putative novel transcripts and for 2) quantifying variation in junction donors and acceptors. We applied SQANTI-reads to two different datasets, a Drosophila developmental experiment and a multi-platform dataset from the LRGASP project and demonstrate that the tool effectively reveals the impact of read coverage on data quality, and readily identifies strong and weak splicing sites. SQANTI-reads is open source and available for download at GitHub.