BACKGROUND Next generation sequencing (NGS) has become a universal practice in modern molecular biology. As the throughput of sequencing experiments increases, the preparation of conventional multiplexed libraries becomes more labor intensive. Conventional library preparation typically requires quality control (QC) testing for individual libraries such as amplification success evaluation and quantification, none of which occur until the end of the library preparation process. RESULTS In this study, we address the need for a more streamlined high-throughput NGS workflow by tethering real-time quantitative PCR (qPCR) to conventional workflows to save time and implement single tube and single reagent QC. We modified two distinct library preparation workflows by replacing PCR and quantification with qPCR using SYBR Green I. qPCR enabled individual library quantification for pooling in a single tube without the need for additional reagents. Additionally, a melting curve analysis was implemented as an intermediate QC test to confirm successful amplification. Sequencing analysis showed comparable percent reads for each indexed library, demonstrating that pooling calculations based on qPCR allow for an even representation of sequencing reads. To aid the modified workflow, a software toolkit was developed and used to generate pooling instructions and analyze qPCR and melting curve data. CONCLUSIONS We successfully applied fluorescent amplification for next generation sequencing (FA-NGS) library preparation to both plasmids and bacterial genomes. As a result of using qPCR for quantification and proceeding directly to library pooling, the modified library preparation workflow has fewer overall steps. Therefore, we speculate that the FA-NGS workflow has less risk of user error. The melting curve analysis provides the necessary QC test to identify and troubleshoot library failures prior to sequencing. While this study demonstrates the value of FA-NGS for plasmid or gDNA libraries, we speculate that its versatility could lead to successful application across other library types.

中文翻译：

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用于下一代测序（FA-NGS）文库制备的荧光扩增。

背景技术下一代测序（NGS）已经成为现代分子生物学中的普遍实践。随着测序实验产量的增加，常规多重文库的制备变得更加费力。传统的文库制备通常需要对单个文库进行质量控制（QC）测试，例如扩增成功率评估和定量，直到文库制备过程结束时才进行。结果在这项研究中，我们通过将实时定量PCR（qPCR）与传统的工作流程绑定在一起，以节省时间并实现单管和单试剂QC，从而满足了对更简化的高通量NGS工作流程的需求。我们通过使用SYBR Green I用qPCR代替PCR和定量来修改两个截然不同的文库制备工作流程。qPCR使单个文库定量成为可能，从而可在单个试管中合并，而无需其他试剂。另外，进行熔解曲线分析作为中间质量控制测试，以确认扩增成功。测序分析显示每个索引文库的读物百分比相当，这表明基于qPCR的合并计算可实现测序读物的均匀表达。为了帮助修改后的工作流程，开发了一个软件工具包，用于生成合并说明并分析qPCR和解链曲线数据。结论我们成功地将荧光扩增用于质粒和细菌基因组的下一代测序（FA-NGS）文库制备。由于使用qPCR进行定量并直接进行库合并，修改后的库准备工作流程的总体步骤更少。因此，我们推测FA-NGS工作流程具有较少的用户错误风险。熔解曲线分析提供了必要的QC测试，以在测序之前识别和排除文库故障。尽管这项研究证明了FA-NGS对于质粒或gDNA文库的价值，但我们推测，它的多功能性可以导致在其他文库类型中的成功应用。