Institute of Molecular Life Sciences, University of Zurich, 8057, Zurich, Switzerland.
SIB Swiss Institute of Bioinformatics, 8057, Zurich, Switzerland.
Nat Commun. 2019 Jul 31;10(1):3359. doi: 10.1038/s41467-019-11272-z.
A platform for highly parallel direct sequencing of native RNA strands was recently described by Oxford Nanopore Technologies, but despite initial efforts it remains crucial to further investigate the technology for quantification of complex transcriptomes. Here we undertake native RNA sequencing of polyA + RNA from two human cell lines, analysing ~5.2 million aligned native RNA reads. To enable informative comparisons, we also perform relevant ONT direct cDNA- and Illumina-sequencing. We find that while native RNA sequencing does enable some of the anticipated advantages, key unexpected aspects currently hamper its performance, most notably the quite frequent inability to obtain full-length transcripts from single reads, as well as difficulties to unambiguously infer their true transcript of origin. While characterising issues that need to be addressed when investigating more complex transcriptomes, our study highlights that with some defined improvements, native RNA sequencing could be an important addition to the mammalian transcriptomics toolbox.
最近，Oxford Nanopore Technologies 公司描述了一种用于高度平行的直接对天然 RNA 链进行测序的平台，但尽管最初做了一些努力，对于复杂转录组的定量分析，仍有必要进一步研究该技术。在这里，我们对来自两种人类细胞系的 polyA+RNA 进行了天然 RNA 测序，分析了约 520 万个对齐的天然 RNA 读段。为了能够进行有意义的比较，我们还进行了相关的 ONT 直接 cDNA 和 Illumina 测序。我们发现，尽管天然 RNA 测序确实能够实现一些预期的优势，但目前关键的意外方面却阻碍了其性能，最明显的是，从单个读段中获得全长转录本的能力相当频繁地受到限制，以及难以明确推断其真实转录本来源的问题。虽然我们的研究强调了在研究更复杂的转录组时需要解决的问题，但当对哺乳动物转录组学工具包进行一些定义的改进时，天然 RNA 测序可能是一个重要的补充。
