16S rRNA Sequencing
16S ribosomal RNA (rRNA) sequencing is an amplicon sequencing technique used to identify and compare species of bacteria present within a given sample. 16S rRNA gene sequencing is used to study phylogeny and taxonomy of samples from complex microbiomes or environments that are difficult or impossible to study. 16S rRNA sequencing can be used to specify taxonomic assignments, down to the species level. Bioo Scientific offers NEXTflex® 16S Amplicon-Seq Kits for this purpose, which are optimized library prep kits developed to simplify 16S rRNA sequencing on the Illumina® MiSeq® sequencing platform. Automation protocols are also now available for the PerkinElmer Sciclone NGS and NGSx Workstation.
Culture-free Identification of Bacteria
The NEXTflex 16S Amplicon-Seq Kits enable analysis of the entire microbial community within a sample, without bias introduced by the culturing required for other, non-NGS based, 16S rRNA analysis methods. With the ability to multiplex hundreds of samples in a sequencing run, NGS-based 16S rRNA sequencing is a sensitive and cost-effective technique to identify strains that may not be identified using other methods.
- Optimized protocol offers lower PCR bias and fewer off-target reads
- Fast library prep protocol
- Low input – As low as 1 ng of genomic DNA
- Flexible barcode options– Up to 384 unique barcodes available for multiplexing of libraries
- Does not require custom sequencing primers
- Automation protocols are now available for the PerkinElmer Sciclone NGS and NGSx Workstation to automate your 16S sequencing
- Functionally validated on the Illumina® MiSeq® sequencer
The NEXTflex™ 16S V1 - V3 Amplicon-Seq Library Prep Kit is designed for the preparation of multiplexed amplicon libraries that span the hypervariable domains one through three (V1-V3) of microbial 16S ribosomal RNA (rRNA) genes. These libraries are compatible with paired-end sequencing on the Illumina® MiSeq® sequencing platform.
Fast Library Prep Protocol
There are two main steps involved in 16S V1-V3 amplicon processing: an initial PCR amplification using customized PCR primers that target the V1-V3 domains, and a subsequent PCR amplification that integrates relevant flow cell binding domains and unique 12 base pair sample indices. The limited number of cleanup steps ensures maximum recovery of amplicons for downstream sequencing.
Optimized Protocol Offers Lower PCR Bias and Fewer Off-target Reads
The protocol incorporated in the NEXTflex 16S V1 - V3 Amplicon-Seq Kit offers better sequencing results than can be obtained using traditional 16S sequencing protocols. The incorporation of the second PCR step in the protocol for the addition of the sample-specific index reduces the number of off-target reads typically encountered during amplicon sequencing.
Selected Publications that Reference using the NEXTflex 16S V1 – V3 Amplicon-Seq Kit:
Maiuri, A. R., et al. (2017) Mismatch Repair Proteins Initiate Epigenetic Alterations during Inflammation-Driven Tumorigenesis. Cancer Research, 77(13), 3467-3478. doi:10.1158/0008-5472.can-17-0056.
Quereda, J. J., et al. (2016) Bacteriocin from epidemic Listeria strains alters the host intestinal microbiota to favor infection. PNAS. doi:10.1073/pnas.1523899113.
Ranjan, R., Rani, A., Metwally, A., McGee, H. S. and Perkins D. L. (2015) Analysis of the microbiome: Advantages of whole genome shotgun versus 16S amplicon sequencing. Biochem Biophy Res Com. doi:10.1016/j.bbrc.2015.12.083.
Yao, J. et al. (2016) A Pathogen-Selective Antibiotic Minimizes Disturbance to the Microbiome. Antimicrob. Agents Chemother. 00535-16. doi:10.1128/AAC.00535-16.