Improved efficiency of two-step amplicon PCR using an acoustic liquid handler

Authors: BENZ, BROOKE - North Dakota State University; LOPES ECHARTEA, EGLATINA - North Dakota State University; Whitaker, Briana; BALDWIN, THOMAS - North Dakota State University; GEDDES, BARNEY - North Dakota State University

Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/13/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: Advances in next generation sequencing technologies over the last 20 years have greatly improved our ability to characterize the microbial communities living on crops. This characterization has shown that microbial communities greatly contribute to overall plant health and productivity. However, while sequencing costs have decreased over time, the cost and time to prepare samples for sequencing have remained largely unchanged. Therefore, ARS researchers in Peoria, Illinois, in collaboration with scientists from North Dakota State University, compared the costs required for traditional sample preparation relative to an automated, high-throughput protocol. The automated protocol was able to reduce sample preparation costs by 30% without affecting the overall sequencing results. Ultimately, the reduction in sample preparation costs will allow researchers worldwide to characterize more crop microbial communities for less, without affecting the quality of their sequencing results.

Technical Abstract: 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 polymerase chain reaction (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 five-fold and increased efficiency for library preparation by ~32% without affecting bacterial community compositions. The associated increase in efficiency of our automated method will allow for greater throughput in sequencing hundreds of microbiome samples without affecting the quality of those sequences.