美国约翰霍普金斯大学Winston Timp和加州大学圣克鲁斯分校Mark Akeson团队合作开发人多聚（A）转录组的纳米孔原位RNA测序技术，这一成果2019年11月18日在线发表在国际学术期刊《自然—方法学》上。

他们使用牛津纳米孔技术公司开发的原位poly（A）RNA测序策略来突破很多局限。该研究使用六个机构的三十个MinION流通池为人类细胞系GM12878生成了990万个比对序列片段。这些原位RNA片段的中位长度为771个碱基，最大比对长度超过21,000个碱基。线粒体poly（A）片段提供了读取长度质量的内部度量。他们将这些长纳米孔片段与更高准确度的短片段和带注释的GM12878启动子区域相结合，以鉴定出33,984个可能的RNA亚型。 他们描述了评估3'poly（A）尾巴长度，碱基修饰和转录单倍型的策略。

据了解，高通量互补DNA测序技术使人类对转录组的复杂性和调控有了更深入的了解。但是，这些方法会丢失生物RNA中包含的信息，因为复制的读段通常很短，并且修饰没有保留。

附：英文原文

Title: Nanopore native RNA sequencing of a human poly(A) transcriptome

Author: Rachael E. Workman, Alison D. Tang, Paul S. Tang, Miten Jain, John R. Tyson, Roham Razaghi, Philip C. Zuzarte, Timothy Gilpatrick, Alexander Payne, Joshua Quick, Norah Sadowski, Nadine Holmes, Jaqueline Goes de Jesus, Karen L. Jones, Cameron M. Soulette, Terrance P. Snutch, Nicholas Loman, Benedict Paten, Matthew Loose, Jared T. Simpson, Hugh E. Olsen, Angela N. Brooks, Mark Akeson, Winston Timp

Issue&Volume: 2019-11-18

Abstract: High-throughput complementary DNA sequencing technologies have advanced our understanding of transcriptome complexity and regulation. However, these methods lose information contained in biological RNA because the copied reads are often short and modifications are not retained. We address these limitations using a native poly(A) RNA sequencing strategy developed by Oxford Nanopore Technologies. Our study generated 9.9 million aligned sequence reads for the human cell line GM12878, using thirty MinION flow cells at six institutions. These native RNA reads had a median length of 771 bases, and a maximum aligned length of over 21,000 bases. Mitochondrial poly(A) reads provided an internal measure of read-length quality. We combined these long nanopore reads with higher accuracy short-reads and annotated GM12878 promoter regions to identify 33,984 plausible RNA isoforms. We describe strategies for assessing 3′ poly(A) tail length, base modifications and transcript haplotypes.

DOI: 10.1038/s41592-019-0617-2

Source: https://www.nature.com/articles/s41592-019-0617-2