Oguchi Yusuke, Shintaku Hirofumi, Uemura Sotaro
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan.
RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan.
Commun Biol. 2020 Dec 18;3(1):788. doi: 10.1038/s42003-020-01499-8.
Single-cell transcriptome analysis has been revolutionized by DNA barcodes that index cDNA libraries, allowing highly multiplexed analyses to be performed. Furthermore, DNA barcodes are being leveraged for spatial transcriptomes. Although spatial resolution relies on methods used to decode DNA barcodes, achieving single-molecule decoding remains a challenge. Here, we developed an in-house sequencing system inspired by a single-molecule sequencing system, HeliScope, to spatially decode DNA barcode molecules at single-molecule resolution. We benchmarked our system with 30 types of DNA barcode molecules and obtained an average read length of ~20 nt with an error rate of less than 5% per nucleotide, which was sufficient to spatially identify them. Additionally, we spatially identified DNA barcode molecules bound to antibodies at single-molecule resolution. Leveraging this, we devised a method, termed "molecular foot printing", showing potential for applying our system not only to spatial transcriptomics, but also to spatial proteomics.
用于索引cDNA文库的DNA条形码彻底改变了单细胞转录组分析，使得高度多重分析得以进行。此外，DNA条形码正被用于空间转录组学。尽管空间分辨率依赖于用于解码DNA条形码的方法，但实现单分子解码仍然是一项挑战。在这里，我们开发了一种受单分子测序系统HeliScope启发的内部测序系统，以单分子分辨率对DNA条形码分子进行空间解码。我们用30种DNA条形码分子对我们的系统进行了基准测试，获得了平均约20 nt的读长，每个核苷酸的错误率低于5%，这足以对它们进行空间识别。此外，我们以单分子分辨率在空间上识别了与抗体结合的DNA条形码分子。利用这一点，我们设计了一种称为“分子足迹”的方法，表明我们的系统不仅在空间转录组学，而且在空间蛋白质组学中都有应用潜力。
