The use of iconPCR for 16S library preparation improves data quality and workflow

Polymerase Chain Reaction (PCR) is a cornerstone of contemporary biological research, enabling the amplification of specific DNA sequences for various applications. However, suboptimal cycling conditions often undermine its efficacy, which can generate chimeric products, exacerbate PCR duplication rates, and skew species representation in metabarcoding experiments due to the preferential amplification of dominant populations. To address these limitations, we present iconPCR--Individually Controlled PCR--a novel technology that allows each reaction in a 96-well plate to be cycled independently and programmatically. By setting a predefined fluorescence threshold, iconPCR ensures that all Next Generation Sequencing (NGS) libraries are amplified to equivalent levels, thereby eliminating the risks of over- or under-amplification through a process known as "Auto-Normalization." In this study, we applied iconPCR to generate V3, V4, and full-length 16S rRNA gene libraries using Avidite sequencing. The V1-V9 variable region libraries were also evaluated with HiFi sequencing, which significantly demonstrated its potential to improve microbial community analysis accuracy and reliability.