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High throughput RNA sequencing technologies have dramatically advanced our understanding of transcriptome complexity and regulation. However, these cDNA-based methods lose information contained in biological RNA because the copied reads are short or because modifications are not carried forward in cDNA. Here we address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies (ONT). Our study focused on poly(A) RNA isolated from the human cell line GM12878, from which we sequenced approximately 9.9 million individual aligned strands. These native RNA sequence reads had an N50 length of 1334 bases, and a maximum length of 22,000 bases. A total of 78,199 high-confidence isoforms were identified by combining long nanopore reads with short higher accuracy Illumina reads. Among these isoforms, over 50% are not present in GENCODE v24. We describe strategies for assessing 3'poly(A) tail length, base modifications and transcript haplotypes using this single molecule technology. Together, these nanopore-based techniques are poised to deliver new insights into RNA biology.

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