Department of Bioengineering and Therapeutic Sciences, California Institute for Quantitative Biosciences (QB3), University of California, San Francisco, California 94158, USA.
UC Berkeley - UCSF Bioengineering Graduate program, University of California, San Francisco, California 94158, USA.
Nat Commun. 2016 Jun 29;7:11784. doi: 10.1038/ncomms11784.
The ability to accurately sequence long DNA molecules is important across biology, but existing sequencers are limited in read length and accuracy. Here, we demonstrate a method to leverage short-read sequencing to obtain long and accurate reads. Using droplet microfluidics, we isolate, amplify, fragment and barcode single DNA molecules in aqueous picolitre droplets, allowing the full-length molecules to be sequenced with multi-fold coverage using short-read sequencing. We show that this approach can provide accurate sequences of up to 10 kb, allowing us to identify rare mutations below the detection limit of conventional sequencing and directly link them into haplotypes. This barcoding methodology can be a powerful tool in sequencing heterogeneous populations such as viruses.
准确测序长 DNA 分子在生物学中非常重要，但现有的测序仪在读取长度和准确性上存在限制。在这里，我们展示了一种利用短读测序来获得长而准确的读段的方法。我们使用液滴微流控技术，在水微微升液滴中分离、扩增、片段化和标记单个 DNA 分子，从而使用短读测序对全长分子进行多倍覆盖测序。我们表明，这种方法可以提供长达 10 kb 的准确序列，使我们能够识别传统测序检测限以下的罕见突变，并直接将其链接成单倍型。这种条形码方法可以成为测序异质群体（如病毒）的有力工具。
