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Published on: November 18, 2014
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TEQUILA-seq: a versatile and low-cost method for targeted long-read RNA sequencing
Feng Wang1, Yang Xu1,2, Robert Wang1,2
1Center for Computational and Genomic Medicine, Children's Hospital of Philadelphia, Philadelphia, PA, USA.
Nature Communications
|August 8, 2023
Summary
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.
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.
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