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Improved genome sequencing using an engineered transposase

机译：使用工程转座酶改善基因组测序

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BackgroundNext-generation sequencing (NGS) has transformed genomic research by reducing turnaround time and cost. However, no major breakthrough has been made in the upstream library preparation methods until the transposase-based Nextera method was invented. Nextera combines DNA fragmentation and barcoding in a single tube reaction and therefore enables a very fast workflow to sequencing-ready DNA libraries within a couple of hours. When compared to the traditional ligation-based methods, transposed-based Nextera has a slight insertion bias. ResultsHere we present the discovery of a mutant transposase (Tn5-059) with a lowered GC insertion bias through protein engineering. We demonstrate Tn5-059 reduces AT dropout and increases uniformity of genome coverage in both bacterial genomes and human genome. We also observe higher library diversity generated by Tn5-059 when compared to Nextera v2 for human exomes, which leads to less sequencing and lower cost per genome. In addition, when used for human exomes, Tn5-059 delivers consistent library insert size over a range of input DNA, allowing up to a tenfold variance from the 50?ng input recommendation. ConclusionsEnhanced DNA input tolerance of Tn5-059 can translate to flexibility and robustness of workflow. DNA input tolerance together with superior uniformity of coverage and lower AT dropouts extend the applications of transposase based library preps. We discuss possible mechanisms of improvements in Tn5-059, and potential advantages of using the new mutant in varieties of applications including microbiome sequencing and chromatin profiling.
机译：背景技术下一代测序（NGS）通过减少周转时间和成本来改变基因组学研究。但是，在发明基于转座酶的Nextera方法之前，上游文库的制备方法尚未取得重大突破。 Nextera在单个试管反应中结合了DNA片段化和条形码功能，因此能够在几个小时内以非常快速的工作流程对准备就绪的DNA库进行测序。与传统的基于连接的方法相比，基于转置的Nextera具有轻微的插入偏差。结果在此我们发现了通过蛋白质工程技术降低了GC插入偏差的突变型转座酶（Tn5-059）。我们证明Tn5-059减少AT脱落并增加细菌基因组和人类基因组中基因组覆盖率的一致性。我们还观察到，与针对人类外显子组的Nextera v2相比，Tn5-059产生的文库多样性更高，这导致更少的测序和每个基因组的成本更低。此外，当用于人类外显子时，Tn5-059在一定范围的输入DNA范围内可提供一致的文库插入物大小，与50 ng输入建议值相比最多可增加十倍。结论增强的Tn5-059 DNA输入耐受性可以转化为工作流程的灵活性和鲁棒性。 DNA输入耐受性以及覆盖范围的均一性和较低的AT缺失率扩展了基于转座酶的文库制备的应用。我们讨论了Tn5-059改善的可能机制，以及在包括微生物组测序和染色质分析在内的各种应用中使用新突变体的潜在优势。

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