16S rRNA Sequencing with Quick-16S NGS Library Prep Kit
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 aims to standardize the library preparation process for 16S rRNA sequencing. Distinguishing features of the kit are described below.
Fastest 16S rRNA Library Prep:
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.
Simple:
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 the Illumina MiSeq without needing additional custom sequencing primers.
Accurate:
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%.
Increased Coverage:
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.
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| Amplicon Size | The size of the 16S V1-V2 region and the 16S V3-V4 region (including primers) is ~350bp and ~460bp, respectively. The final amplicon size after addition of barcoded primers is ~486bp and ~596bp, respectively. |
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| Barcode Sequences | Available here |
| Equipment | Microcentrifuge, plate spinner (centrifuge), 96-well real-time quantitative PCR system, 96-well real-time PCR plates. |
| Index Primers | Dual index (barcodes) to uniquely label 96 samples. |
| Preparing > 96 Samples | For guidelines to preparing more than 96 samples, please click here. |
| Recommended RT PCR System | Bio-Rad CFX96 Real-Time PCR Detection System (any model), Applied Biosystems 7500 Real-Time PCR System. |
| Sample Input | Purified microbial DNA ≤ 20 ng/µl, free of PCR inhibitors. |
| Sequencing Compatibility | Illumina MiSeq without the need to add custom sequencing primers. Zymo Research recommends the MiSeq Reagent Kit v3 (600-cycle). |
| Supplemental Info |
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