Detection of low-frequency mutations in cancer genomes or other heterogeneous cell populations requires high-fidelity sequencing. Molecular barcoding is one of the key technologies that enables the differentiation of true mutations from errors, which can be caused by sequencing or library preparation processes. However, current approaches where barcodes are introduced via primer extension or adaptor ligation do not utilize the full power of barcoding, due to complicated library preparation workflows and biases. Here we demonstrate the remarkable tolerance of MuA transposase to the presence of multiple replacements in transposon sequence, and explore this unique feature to engineer the MuA transposome complex with randomised nucleotides in 12 transposon positions, which can be introduced as a barcode into the target molecule after transposition event. We applied the approach of Unique MuA-based Molecular Indexing (UMAMI) to assess the power of rare mutation detection by shortgun sequencing on the Illumina platform. Our results show that UMAMI allows detection of rare mutations readily and reliably, and in this paper we report error rate values for the number of thermophilic DNA polymerases measured by using UMAMI.

检测癌症基因组或其他异质细胞群中的低频突变需要高保真测序。分子条形码是一种关键技术，可以区分真正的突变和错误，错误可能是由测序或文库制备过程引起的。然而，由于复杂的文库制备工作流程和偏差，目前通过引物延伸或接头连接引入条形码的方法没有充分利用条形码的功能。在这里，我们证明了 MuA 转座酶对转座子序列中存在多个替换的显着耐受性，并探索了这一独特的特征，以在 12 个转座子位置设计具有随机核苷酸的 MuA 转座体复合物，它可以在转座事件后作为条形码引入目标分子。我们应用了基于独特 MuA 的分子索引 (UMAMI) 的方法来评估在 Illumina 平台上通过短枪测序检测稀有突变的能力。我们的结果表明 UMAMI 可以轻松可靠地检测稀有突变，在本文中，我们报告了使用 UMAMI 测量的嗜热 DNA 聚合酶数量的错误率值。