Benz Brooke R, Lopez-Echartea Eglantina, Whitaker Briana K, Baldwin Thomas, Geddes Barney A
Department of Plant Pathology, North Dakota State University, Fargo, ND, USA.
Department of Microbiological Sciences, North Dakota State University, Fargo, ND, USA.
Microbiology (Reading). 2025 Jul;171(7). doi: 10.1099/mic.0.001579.
The improvement in next-generation sequencing technologies has reduced the costs of sequencing significantly. However, library preparation costs for amplicon sequencing have remained largely unchanged - which is ultimately the cost-limiting step in processing large numbers of microbiome samples. Acoustic liquid handlers can transfer volumes as low as 2.5 nl and have been used to miniaturize several different molecular and cellular assays, including single-step PCR amplicon library preparations. However, there are no current methods available for a two-step library preparation process using an acoustic liquid handler. In this study, we tested the efficiency of an acoustic liquid handler to automate the PCRs and library quantification while also incorporating automated library bead cleanup. We compared the material usage and costs for library preparation and sequencing results of this automated method to the standard, manual method. The automated protocol was able to reduce both PCR reaction volumes fivefold and increased efficiency for library preparation by ~32% without affecting bacterial community compositions. The associated increase in the efficiency of our automated method will allow for greater throughput in sequencing hundreds of microbiome samples without affecting the quality of those sequences.
下一代测序技术的改进显著降低了测序成本。然而，扩增子测序的文库制备成本在很大程度上保持不变——这最终是处理大量微生物组样本的成本限制步骤。声学液体处理仪可以转移低至2.5 nl的体积，并已用于小型化几种不同的分子和细胞检测，包括单步PCR扩增子文库制备。然而，目前没有使用声学液体处理仪进行两步文库制备过程的方法。在本研究中，我们测试了声学液体处理仪在自动化PCR和文库定量同时结合自动文库磁珠纯化的效率。我们将这种自动化方法的文库制备材料使用量和成本以及测序结果与标准手动方法进行了比较。自动化方案能够将PCR反应体积减少五倍，并将文库制备效率提高约32%，同时不影响细菌群落组成。我们自动化方法效率的相关提高将允许在不影响数百个微生物组样本测序质量的情况下实现更高的通量。
