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Evaluating the potential and limitations of nanopore adaptive sampling for targeted transcriptome sequencing
Nicole DeBruyne1, Feng Wang2, Yang Xu2,3
1Graduate Group in Cell and Molecular Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA.
Genome Biology
|October 10, 2025
Summary
Adaptive sampling in long-read RNA sequencing modestly enriches targets but is less effective than hybridization capture. Direct RNA sequencing shows potential for boosting yield in time-sensitive applications.
Area of Science:
- Genomics
- Molecular Biology
- Bioinformatics
Background:
- Long-read RNA sequencing offers comprehensive transcriptomic insights.
- High cost and low throughput limit its widespread application.
- Adaptive sampling is an enrichment strategy for nanopore sequencing.
Purpose of the Study:
- To evaluate the efficacy of adaptive sampling for human transcriptome analysis.
- To compare adaptive sampling with traditional cDNA hybridization capture.
- To assess the impact of adaptive sampling on gene expression and splicing data.
Main Methods:
- Utilized Oxford Nanopore Technologies adaptive sampling.
- Performed both cDNA and direct RNA sequencing.
- Analyzed human transcriptome data, comparing enrichment efficiency and data quality.
Main Results:
- Adaptive sampling provided modest enrichment (1.3x for cDNA, 1.9x for direct RNA).
- Gene expression and splicing profiles were preserved.
- Effectiveness was significantly lower than cDNA hybridization capture.
- Short read lengths and low sequencing quality impacted performance.
Conclusions:
- Adaptive sampling offers a moderate enrichment strategy for long-read RNA sequencing.
- Direct RNA sequencing with adaptive sampling can increase target yield in fixed run times.
- Further improvements in sequencing technology are needed to maximize adaptive sampling benefits.
