16S rRNA sequencing is a routine technique for microbiome composition profiling. Compared to shotgun metagenomics sequencing, 16S rRNA sequencing is more cost-effective and more robust; it generally requires less input DNA and is less impacted by the presence of host DNA. However, 16S rRNA sequencing has its own challenges. One major challenge is the formation of PCR chimeric sequences, which are artificial sequences resulting from the recombination of two or more PCR templates. Additionally, with common 16S primers, it is difficult to achieve both species-level resolution and broad phylogenetic coverage. Moreover, common 16S library preparation protocols used in the field have not been optimized to be cost-effective for large-scale applications.

The Quick-16S™ NGS Library Prep Kit utilizes real-time (quantitative) PCR (qPCR) rather than endpoint PCR for 16S rRNA amplification, enabling direct quantification of PCR products and eliminating the need for additional library quantification analysis such as TapeStation®analysis or gel electrophoresis. An enzymatic clean-up is introduced between the two PCR steps, saving time and reducing costs as compared to lengthy AMPure®bead-based clean-ups. With these features, the kit dramatically reduces the hands-on time of 16S library preparation. The Quick-16S™ NGS Library Prep Kit includes all the reagents needed to convert 96 DNA samples to a 16S library. The resulting library is directly compatible with Illumina MiSeq®without needing additional custom sequencing primers. The utilization of real-time PCR also enables users to control PCR cycles. This limits chimera formation and PCR bias while obtaining enough products for subsequent sequencing. In most cases, the abundance of PCR chimeric sequences is maintained below 2%. Due to the rapid expansion of 16S rRNA databases, the insufficient microbial coverage of common 16S primer sets has become evident. Zymo Research has re-designed two common primer sets targeting the 16S V1-V2 and 16S V3-V4 regions based on the most updated 16S reference database and significantly improved their coverage.