Abstract
The poly(A) tail is a homopolymeric stretch of adenosine at the 3′-end of mature RNA transcripts and its length plays an important role in nuclear export, stability, and translational regulation of mRNA. Existing techniques for genome-wide estimation of poly(A) tail length are based on short-read sequencing. These methods are limited because they sequence a synthetic DNA copy of mRNA instead of the native transcripts. Furthermore, they can identify only a short segment of the transcript proximal to the poly(A) tail which makes it difficult to assign the measured poly(A) length uniquely to a single transcript isoform. With the introduction of native RNA sequencing by Oxford Nanopore Technologies, it is now possible to sequence full-length native RNA. A single long read contains both the transcript and the associated poly(A) tail, thereby making transcriptome-wide isoform-specific poly(A) tail length assessment feasible. We developed tailfindr—an R-based package for estimating poly(A) tail length from Oxford Nanopore sequencing data. In this chapter, we describe in detail the pipeline for transcript isoform-specific poly(A) tail profiling based on native RNA Nanopore sequencing—from library preparation to downstream data analysis with tailfindr.
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Adnan M. Niazi and Maximilian Krause contributed equally to this work.
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Niazi, A.M., Krause, M., Valen, E. (2021). Transcript Isoform-Specific Estimation of Poly(A) Tail Length by Nanopore Sequencing of Native RNA. In: Picardi, E. (eds) RNA Bioinformatics. Methods in Molecular Biology, vol 2284. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1307-8_30
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DOI: https://doi.org/10.1007/978-1-0716-1307-8_30
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