Adaptive sampling for nanopore direct RNA-sequencing.

Autor:	Naarmann-de Vries IS; Klaus Tschira Institute for Integrative Computational Cardiology, University Hospital Heidelberg, 69120 Heidelberg, Germany.; German Center for Cardiovascular Research (DZHK), Partner site Heidelberg/Mannheim, 69120 Heidelberg, Germany., Gjerga E; Klaus Tschira Institute for Integrative Computational Cardiology, University Hospital Heidelberg, 69120 Heidelberg, Germany.; German Center for Cardiovascular Research (DZHK), Partner site Heidelberg/Mannheim, 69120 Heidelberg, Germany., Gandor CLA; Klaus Tschira Institute for Integrative Computational Cardiology, University Hospital Heidelberg, 69120 Heidelberg, Germany., Dieterich C; Klaus Tschira Institute for Integrative Computational Cardiology, University Hospital Heidelberg, 69120 Heidelberg, Germany christoph.dieterich@uni-heidelberg.de.; German Center for Cardiovascular Research (DZHK), Partner site Heidelberg/Mannheim, 69120 Heidelberg, Germany.
Jazyk:	angličtina
Zdroj:	RNA (New York, N.Y.) [RNA] 2023 Dec; Vol. 29 (12), pp. 1939-1949. Date of Electronic Publication: 2023 Sep 06.
DOI:	10.1261/rna.079727.123
Abstrakt:	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. (© 2023 Naarmann-de Vries et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
Databáze:	MEDLINE
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