Naarmann-de Vries, I. S. | bioRxiv Science

bioRxiv2022

Adaptive Sampling as tool for Nanopore direct RNA-sequencing

ONT long-read sequencing provides real-time monitoring and controlling of individual nanopores. Adaptive sampling enriches or depletes specific sequences in Nanopore DNA sequencing, but was not applicable to direct sequencing of RNA so far. Here, we identify essential parameter settings for direct RNA sequencing (DRS). We demonstrate the superior performance of depletion over enrichment and show that adaptive sampling efficiently depletes specific transcripts in transcriptome-wide sequencing applications. Specifically, we applied our adaptive sampling approach to polyA+ RNA samples from human cardiomyocytes and mouse whole heart tissue. Herein, we show more than 2.5-fold depletion of highly abundant mitochondrial-encoded transcripts that in normal sequencing account for up to 40% of sequenced bases in heart tissue samples.

bioinformatics↗

bioRxiv2021

Deep assessment of human disease-associated ribosomal RNA modifications using Nanopore direct RNA sequencing

The catalytically active component of ribosomes, rRNA, is long studied and heavily modified. However, little is known about functional and pathological consequences of changes in human rRNA modification status. Direct RNA sequencing on the Nanopore platform enables the direct assessment of rRNA modifications. We established a targeted Nanopore direct rRNA sequencing approach and applied it to CRISPR-Cas9 engineered HCT116 cells, lacking specific enzymatic activities required to establish defined rRNA base modifications. We analyzed these sequencing data along with wild type samples and in vitro transcribed reference sequences to specifically detect changes in modification status. We show for the first time that direct RNA-sequencing is feasible on smaller, i.e. Flongle, flow cells. Our targeted approach reduces RNA input requirements, making it accessible to the analysis of limited samples such as patient derived material. The analysis of rRNA modifications during cardiomyocyte differentiation of human induced pluripotent stem cells, and of heart biopsies from cardiomyopathy patients revealed altered modifications of specific sites, among them pseudouridines, 2-O-methylation of riboses and acetylation of cytidines. Targeted direct rRNA-seq analysis with JACUSA2 opens up the possibility to analyze dynamic changes in rRNA modifications in a wide range of biological and clinical samples.

molecular biology↗