High throughput error corrected Nanopore single cell transcriptome sequencing
High throughput error corrected Nanopore single cell transcriptome sequencing
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DOI：
10.1038/s41467-020-17800-6
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发表时间：
2020-08-12
影响因子：
16.6
通讯作者：
Waldmann, Rainer
中科院分区：
文献类型：
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作者：
Lebrigand, Kevin;Magnone, Virginie;Waldmann, Rainer
基于液滴的高通量单细胞测序技术极大地提高了我们对细胞间异质性的认识。然而，这些方法只允许在短读测序后分析转录本的一个末端。结果，关于剪接和序列异质性的信息丢失了。为了克服这一限制，最近引入了几种使用长读测序的方法。然而，这些技术受到测序深度低和/或缺乏或不准确的唯一分子标识符（UMIs）分配的限制，这对于消除PCR偏差和伪影至关重要。我们介绍了ScNaUmi-seq，一种将牛津纳米孔测序的高通量与精确的细胞条形码和UMI分配策略相结合的方法。UMI引导的纠错允许在高测序深度与10x Genomics单细胞分离系统产生高精度的全长序列信息。我们分析了胚胎小鼠大脑的转录异构体多样性，并表明ScNaUmi-seq允许在单细胞水平上定义剪接和snv （RNA编辑）。基于液滴的高通量单细胞测序技术往往会丢失转录物剪接和异质性的信息。在这里，作者介绍了ScNaUmi-seq，它使用牛津纳米孔测序和条形码来生成高精度的全长序列。
Droplet-based high throughput single cell sequencing techniques tremendously advanced our insight into cell-to-cell heterogeneity. However, those approaches only allow analysis of one extremity of the transcript after short read sequencing. In consequence, information on splicing and sequence heterogeneity is lost. To overcome this limitation, several approaches that use long-read sequencing were introduced recently. Yet, those techniques are limited by low sequencing depth and/or lacking or inaccurate assignment of unique molecular identifiers (UMIs), which are critical for elimination of PCR bias and artifacts. We introduce ScNaUmi-seq, an approach that combines the high throughput of Oxford Nanopore sequencing with an accurate cell barcode and UMI assignment strategy. UMI guided error correction allows to generate high accuracy full length sequence information with the 10x Genomics single cell isolation system at high sequencing depths. We analyzed transcript isoform diversity in embryonic mouse brain and show that ScNaUmi-seq allows defining splicing and SNVs (RNA editing) at a single cell level. Droplet-based high throughput single cell sequencing techniques can often lose information on transcript splicing and heterogenity. Here the authors introduce ScNaUmi-seq, which uses Oxford Nanopore sequencing and barcoding to generate high accuracy full length sequences.