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TEQUILA-seq: a versatile and low-cost method for targeted long-read RNA sequencing.

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TEQUILA-seq enhances targeted long-read RNA sequencing for deeper transcript coverage. This cost-effective method reveals novel isoforms and RNA-associated mechanisms for tumor suppressor gene inactivation in cancer.

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Area of Science:

  • Genomics and Transcriptomics
  • Molecular Biology
  • Cancer Research

Background:

  • Long-read RNA sequencing (RNA-seq) offers comprehensive transcriptome analysis but faces limitations in throughput and transcript coverage.
  • Targeted RNA-seq strategies are crucial for overcoming throughput bottlenecks and focusing on specific gene panels.
  • Current methods for targeted long-read RNA-seq can be costly and complex to implement.

Purpose of the Study:

  • To introduce TEQUILA-seq, a novel, cost-effective, and user-friendly method for targeted long-read RNA sequencing.
  • To assess the ability of TEQUILA-seq to enrich transcript coverage and preserve quantification for targeted gene panels.
  • To investigate full-length transcript isoforms of actionable cancer genes in breast cancer cell lines and identify novel isoforms and regulatory mechanisms.

Main Methods:

  • Development of TEQUILA-seq, a targeted long-read RNA-seq method utilizing isothermally linear-amplified capture probes.
  • Application of TEQUILA-seq on the Oxford Nanopore platform with various gene panel sizes.
  • Profiling of full-length transcript isoforms for 468 actionable cancer genes across 40 breast cancer cell lines.

Main Results:

  • TEQUILA-seq consistently and substantially enriched transcript coverage while maintaining accurate quantification across different gene panels.
  • Identification of transcript isoforms specific to breast cancer subtypes and discovery of novel isoforms in genes like TP53.
  • Significant enrichment of aberrant transcript isoforms targeted for degradation via nonsense-mediated decay in tumor suppressor genes, indicating a common inactivation mechanism.

Conclusions:

  • TEQUILA-seq offers a versatile, low-cost, and efficient solution for targeted long-read RNA sequencing, significantly reducing costs compared to commercial alternatives.
  • The method enables deep profiling of full-length transcript isoforms, facilitating the discovery of novel isoforms and regulatory mechanisms in cancer.
  • TEQUILA-seq has broad applicability in biomedical research for targeted sequencing of full-length transcripts.