Improved Efficiency of Two-Step Amplicon PCR Using an Acoustic Liquid Handler

Abstract

2.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. 3. Impact statementLibrary preparation for amplicon sequencing in microbiome studies remains a significant cost constraint, even while sequencing costs have decreased in the last few decades. Acoustic liquid handling robots have been utilized to miniaturize several different assays, including a one-step PCR library preparation process, to reduce costs. However, there are currently no methods available for utilizing acoustic liquid handlers for two-step PCR library preparation. Here, we show that the incorporation of an acoustic liquid handler and automated purification instruments into a two-step library preparation protocol led to a reduction in costs and time to prepare libraries. This method is comparable to the standard manual method and will lead to significant cost savings for the preparation of hundreds of samples required for microbiome studies. 4. Data summaryAll protocols, sequence data, and analysis codes have been made publicly available in open-access repositories. The authors confirm all supporting data, code, and protocols have been provided within the article or through supplementary data files. Sequencing data was deposited in the NCBI SRA under the BioProject PRJNA1190462. R scripts used for data analysis are available at https://github.com/NDSU-Geddes-Lab/brb-libprep doi: 10.5281/zenodo.14194699. Protocols for automated library preparation have been published on protocols.io and are available at dx.doi.org/10.17504/protocols.io.e6nvwb5r2vmk/v2 and dx.doi.org/10.17504/protocols.io.e6nvwb5w2vmk/v2.