Nanopore long-read sequencing enables real-time monitoring and controlling of individual nanopores. This allows us to enrich or deplete specific sequences in DNA sequencing in a process called “adaptive sampling.” So far, adaptive sampling (AS) was not applicable to the direct sequencing of RNA. Here, we show that AS is feasible and useful for direct RNA sequencing (DRS), which has its specific technical and biological challenges. Using a well-controlled in vitro transcript-based model system, we identify essential characteristics and parameter settings for AS in DRS, as the superior performance of depletion over enrichment. Here, the efficiency of depletion is close to the theoretical maximum. Additionally, we demonstrate that AS efficiently depletes specific transcripts in transcriptome-wide sequencing applications. Specifically, we applied our AS approach to poly(A)-enriched RNA samples from human-induced pluripotent stem cell–derived cardiomyocytes and mouse whole heart tissue and show efficient 2.5- to 2.8-fold depletion of highly abundant mitochondrial-encoded transcripts. Finally, we characterize depletion and enrichment performance for complex transcriptome subsets, that is, at the level of the entire Chromosome 11, proving the general applicability of direct RNA AS. Our analyses provide evidence that AS is especially useful to enable the detection of lowly expressed transcripts and reduce the sequencing of highly abundant disturbing transcripts.

中文翻译：

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纳米孔直接 RNA 测序的自适应采样

纳米孔长读长测序能够实时监测和控制单个纳米孔。这使我们能够在称为“自适应采样”的过程中丰富或消除 DNA 测序中的特定序列。迄今为止，自适应采样（AS）还不适用于RNA的直接测序。在这里，我们证明 AS 对于直接 RNA 测序 (DRS) 是可行且有用的，但它有其特定的技术和生物学挑战。使用良好控制的体外基于转录的模型系统，我们确定了 DRS 中 AS 的基本特征和参数设置，因为耗尽相对于富集具有优越的性能。这里，耗尽效率接近理论最大值。此外，我们证明 AS 在全转录组测序应用中有效地消耗特定转录本。具体来说，我们将我们的 AS 方法应用于来自人诱导多能干细胞来源的心肌细胞和小鼠全心脏组织的富含聚腺苷酸的 RNA 样本，并显示出高丰度线粒体编码转录本的有效去除 2.5 至 2.8 倍。最后，我们描述了复杂转录组子集（即整个 11 号染色体水平）的耗尽和富集性能，证明了直接 RNA AS 的普遍适用性。我们的分析提供的证据表明，AS 对于检测低表达转录本和减少高丰度干扰转录本的测序特别有用。