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Using polyinosine tailing to capture RNA 3' ends by Direct-RNA Nanopore sequencing

Creative Commons 'BY-NC-ND' version 4.0 license
Abstract

Transcriptome is a word that describes the information contained in all the RNAs in a cell or cell population. Capturing the transcriptome requires RNA sequencing, giving a snapshot on the biological processes happening in the organism or cell. A popular sequencing platform for sequencing the transcriptome is Next Generation Sequencing (NGS) from Illumina, a short-read, RT-PCR based system that sequences RNA sample through amplified cDNA which can cause misrepresentation of the starting RNA population. An alternative to NGS is Direct-RNA Nanopore Sequencing from Oxford Nanopore Technologies (ONT), a long-read sequencing platform that directly sequences the entire length of the RNA transcript. The standard library preparation protocol for direct RNA sequencing is focused on capturing RNAs containing a poly(A) tail. Sequencing of non-poly(A) RNAs is possible through use of custom adaptors designed for a conserved 3' sequence, which limits the population of RNAs that can be sequenced in a sample or by adding poly(A) using poly(A) polymerase which confounds the native 3' end sequence of RNAs. Here, I describe a new technique for Direct-RNA Nanopore sequencing, which modifies the 3′ ends of RNAs with inosine-tails allowing for sequencing of diverse RNA samples. My work demonstrates the versatility and potential of applying the poly(I) sequencing method to diverse transcriptomes, providing a new sequencing method that may enable new RNA discoveries.

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