VIsoQLR: an interactive tool for the detection, quantification and fine-tuning of isoforms in selected genes using long-read sequencing

Núñez‐Moreno, Gonzalo; Tamayo, Alejandra; Ruiz-Sánchez, Carolina; Cortón, Marta; Mínguez, Pablo

doi:10.1007/s00439-023-02539-z

Cited by 2 publications

(1 citation statement)

References 42 publications

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“…Sashimi plots were generated using ggsashimi (36). BAM files were loaded into VIsoQLR (37), which was used to detect and quantify splice isoform species using standard parameters, except for adjustments of the base-pair lengths made to the detected terminal exons to compensate for sequencing biases.…”

Section: Long-range Sequencingmentioning

confidence: 99%

A humanized knock-inCol6a1mouse recapitulates a deep-intronic splice-activating variant

Bolduc,

Guirguis,

Lubben

et al. 2024

Preprint

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Antisense therapeutics such as splice-modulating antisense oligonucleotides (ASOs) are promising tools to treat diseases caused by splice-altering intronic variants. However, their testing in animal models is hampered by the generally poor sequence conservation of the intervening sequences between human and other species. Here we aimed to model in the mouse a recurrent, deep-intronic, splice-activating,COL6A1variant, associated with a severe form of Collagen VI-related muscular dystrophies (COL6-RDs), for the purpose of testing human-ready antisense therapeuticsin vivo. The variant, c.930+189C>T, creates a donor splice site and inserts a 72-nt-long pseudoexon, which, when translated, acts in a dominant-negative manner, but which can be skipped with ASOs. We created a unique humanized mouse allele (designated as ″h″), in which a 1.9 kb of the mouse genomic region encoding the amino-terminus (N-) of the triple helical (TH) domain of collagen α1(VI) was swapped for the human orthologous sequence. In addition, we also created an allele that carries the c.930+189C>T variant on the same humanized knock-in sequence (designated as ″h+189T″). We show that in both models, the human exons are spliced seamlessly with the mouse exons to generate a chimeric mouse-human collagen α1(VI) protein. In homozygousCol6a1h+189T/ h+189Tmice, the pseudoexon is expressed at levels comparable to those observed in heterozygous patients′ muscle biopsies. WhileCol6a1h/hmice do not show any phenotype compared to wild-type animals,Col6a1h/ h+189TandCol6a1h+189T/ h+189Tmice have smaller muscle masses and display grip strength deficits detectable as early as 4 weeks of age. The pathogenic h+189T humanized knock-in mouse allele thus recapitulates the pathogenic splicing defects seen in patients′ biopsies and allows testing of human-ready precision antisense therapeutics aimed at skipping the pseudoexon. Given that theCOL6A1N-TH region is a hot-spot for COL6-RD variants, the humanized knock-in mouse model can be utilized as a template to introduce otherCOL6A1pathogenic variants. This unique humanized mouse model thus represents a valuable tool for the development of antisense therapeutics for COL6-RD.

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Section: Long-range Sequencingmentioning

confidence: 99%

A humanized knock-inCol6a1mouse recapitulates a deep-intronic splice-activating variant

Bolduc,

Guirguis,

Lubben

et al. 2024

Preprint

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Enhancing novel isoform discovery: leveraging nanopore long-read sequencing and machine learning approaches

Santucci,

Cheng,

et al. 2024

Briefings in Functional Genomics

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Long-read sequencing technologies can capture entire RNA transcripts in a single sequencing read, reducing the ambiguity in constructing and quantifying transcript models in comparison to more common and earlier methods, such as short-read sequencing. Recent improvements in the accuracy of long-read sequencing technologies have expanded the scope for novel splice isoform detection and have also enabled a far more accurate reconstruction of complex splicing patterns and transcriptomes. Additionally, the incorporation and advancements of machine learning and deep learning algorithms in bioinformatic software have significantly improved the reliability of long-read sequencing transcriptomic studies. However, there is a lack of consensus on what bioinformatic tools and pipelines produce the most precise and consistent results. Thus, this review aims to discuss and compare the performance of available methods for novel isoform discovery with long-read sequencing technologies, with 25 tools being presented. Furthermore, this review intends to demonstrate the need for developing standard analytical pipelines, tools, and transcript model conventions for novel isoform discovery and transcriptomic studies.

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VIsoQLR: an interactive tool for the detection, quantification and fine-tuning of isoforms in selected genes using long-read sequencing

Cited by 2 publications

References 42 publications

A humanized knock-inCol6a1mouse recapitulates a deep-intronic splice-activating variant

A humanized knock-inCol6a1mouse recapitulates a deep-intronic splice-activating variant

Enhancing novel isoform discovery: leveraging nanopore long-read sequencing and machine learning approaches

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