Quick-16S NGS Library Prep Kit
Quick-16S NGS Library Prep Kit
- Fastest: Only 1.5 hours of hands-on time. No Tapestation® analysis or AMPure® clean-ups.
- Accurate: Utilization of real-time PCR limits PCR chimera formation.
- Increased Coverage: Novel primers increase phylogenetic coverage of Bacteria and Archaea and enable species-level resolution for human microbiome profiling.
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.
|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.|
|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).|
The authors investigated the microbial pools associated with landfill reactors that are impacted by nanomaterials. By preparing 16S rRNA libraries with the Quick-16S NGS Library Prep Kit, they discovered that the bacterial communities were similar between control reactors and nanomaterial-containing reactors, but the archaea communities were highly different.Akyol, Ç., Ozbayram, E.G., Demirel, B., Onay, T.T., Ince, O., and Ince, B. (2019). Linking nano-ZnO contamination to microbial community profiling in sanitary landfill simulations. Environ Sci Pollut Res. doi: 10.1007/s11356-019-04906-8
This study investigated how varying interchange ratios in wastewater treatment affects microbial community composition. Analysis of samples prepared for 16S sequencing with the Quick-16S NGS Library Prep Kit demonstrated that the relative abundance of the phyla Proteobacteria, Acidobacteria, and Bacteroidetes varied according to the interchange ratio and that the species of Thiothrix were highly abundance in all samples, revealing their potential importance to wastewater treatment processes.Karlikanovaite-Balikci, A., Ozbayram, E.G., Yagci, N., and Ince, O. (2019). Microbial community shifts in the oxic-settling-anoxic process in response to changes to sludge interchange ratio. Heliyon, 5(4), e01517. doi: 10.1016/j.heliyon.2019.e01517
Akyol et al. performed 16S and 18S analysis of anaerobic digestates using the Quick-16S NGS Library Prep Kit to assess microbial diversity in lignocellulose-rich digestates. The most abundant bacterial and fungal genera were consistent across digestate samples.Akyol, Ç., Ince, O., and Ince, B. (2019). Crop-based composting of lignocellulosic digestates: Focus on bacterial and fungal diversity. Bioresour. Technol., 121549. doi: 10.1016/j.biortech.2019.121549
“This is a very good kit for library preparation, especially compared to other library preparation products. We love that all the reagents we need to prepare a targeted sequencing library are included!”
- Leyao W. (Washington University in St. Louis)
"We first tested this kit during the beta test in 2018. We really liked it an immediately ordered full kits to change our workflow."
- Lisa D. (University of Hohenheim)
"This is an excellent kit and I highly recommend it."
- Karina S. (Universidad Nacional de Misiones)