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

Feng Wang, Yang Xu, Robert Wang, Beatrice Zhang, Noah Smith, Amber Notaro, Samantha Gaerlan, Eric Kutschera, Kathryn E. Kadash-Edmondson, Yi Xing () and Lan Lin ()
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Feng Wang: Children’s Hospital of Philadelphia
Yang Xu: Children’s Hospital of Philadelphia
Robert Wang: Children’s Hospital of Philadelphia
Beatrice Zhang: Children’s Hospital of Philadelphia
Noah Smith: Children’s Hospital of Philadelphia
Amber Notaro: Children’s Hospital of Philadelphia
Samantha Gaerlan: Children’s Hospital of Philadelphia
Eric Kutschera: Children’s Hospital of Philadelphia
Kathryn E. Kadash-Edmondson: Children’s Hospital of Philadelphia
Yi Xing: Children’s Hospital of Philadelphia
Lan Lin: University of Pennsylvania Perelman School of Medicine

Nature Communications, 2023, vol. 14, issue 1, 1-15

Abstract: Abstract Long-read RNA sequencing (RNA-seq) is a powerful technology for transcriptome analysis, but the relatively low throughput of current long-read sequencing platforms limits transcript coverage. One strategy for overcoming this bottleneck is targeted long-read RNA-seq for preselected gene panels. We present TEQUILA-seq, a versatile, easy-to-implement, and low-cost method for targeted long-read RNA-seq utilizing isothermally linear-amplified capture probes. When performed on the Oxford nanopore platform with multiple gene panels of varying sizes, TEQUILA-seq consistently and substantially enriches transcript coverage while preserving transcript quantification. We profile full-length transcript isoforms of 468 actionable cancer genes across 40 representative breast cancer cell lines. We identify transcript isoforms enriched in specific subtypes and discover novel transcript isoforms in extensively studied cancer genes such as TP53. Among cancer genes, tumor suppressor genes (TSGs) are significantly enriched for aberrant transcript isoforms targeted for degradation via mRNA nonsense-mediated decay, revealing a common RNA-associated mechanism for TSG inactivation. TEQUILA-seq reduces the per-reaction cost of targeted capture by 2-3 orders of magnitude, as compared to a standard commercial solution. TEQUILA-seq can be broadly used for targeted sequencing of full-length transcripts in diverse biomedical research settings.

Date: 2023
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DOI: 10.1038/s41467-023-40083-6

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