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.
NEXTflex® 16S V5 – V6 Amplicon-Seq Kit for Illumina® Library Prep
Validated library prep for bacterial profiling of plant samples using Illumina® sequencing platforms
- Fast and easy library prep protocol for 16S profiling of bacteria in plant samples
- Low input - Only 1 ng of input required
- Multiplex up to 384 libraries
- Custom sequencing primers are not required
- Automation protocols are now available for the PerkinElmer® Sciclone® NGS and NGSx Workstations to automate your 16S sequencing
- Functionally validated with Illumina® sequencing platforms
The NEXTflex™ 16S V5 – V6 Amplicon-Seq Library Prep Kit is designed for bacterial profiling of plant samples using degenerate primers that target the fifth and sixth variable regions of the 16S rRNA gene. Bacterial profiling in plant samples can be difficult because of the homology between bacterial 16S rRNA and chloroplast 16S rRNA. The use of primers binding to 16S hypervariable regions V 1 – V4, which are traditionally used for bacterial profiling, amplify both the bacterial and plant 16S rRNA genes, skewing results. NEXTflex™ 16S V5 – V6 Amplicon-Seq libraries are compatible with paired-end sequencing on the Illumina® MiSeq® sequencing platform and are ideal for monitoring population fluctuations of microbial communities.
Difference in Representation between 16S V4 and 16S V5-V6 in the Presence of Plant Contamination
The difference between the 0% and 90% plant-contaminated samples was calculated for each species and each primer set. The 16S V4 amplicons show significant variability in the representation of bacterial community members with increasing plant DNA contamination. In contrast, the 16S V5-V6 amplicons show very little sensitivity to the addition of up to 90% of plant DNA.
Optimized Protocol Offers Lower PCR Bias and Fewer Off-target Reads
The NEXTflex 16S V5 – V6 Amplicon-Seq Kit incorporates a second PCR step in the protocol for the addition of the sample-specific index, which reduces off-target reads encountered during amplicon sequencing.
Low Input Requirements for 16S rRNA Sequencing
As little as 1 ng of genomic DNA can be used to generate libraries using the NEXTflex 16S V5 – V6 Amplicon-Seq Library Prep Kit.
384 Barcodes Available for Cost-effective 16S V5 – V6 rRNA Sequencing
The NEXTflex 16S V5 – V6 Amplicon-Seq Library Prep Kit can be used for multiplexing up to 384 samples, to greatly reduce the cost of sequencing. The barcodes are offered in sets of 4, 12, 48, or 96 barcodes and supplied with 2 reactions worth of each barcode.
For Research Use Only. Not for use in diagnostic procedures.
|NEXTflex® 16S V5 - V6 Amplicon-Seq Kit (48 Barcodes)
Automation Next Gen Sequencing
The NEXTflex 16S V5 – V6 Amplicon-Seq Kit contains enough material to prepare 8, 24, 96, or 192 amplicon-seq libraries from bacterial genomic DNA for sequencing using the Illumina® MiSeq® platform. The shelf life of all reagents is 12 months when stored properly. All components can be safely stored at -20°C. This kit is shipped on dry ice.
NEXTflex 16S V5 – V6 Amplicon-Seq Kit Protocol
- NEXTflex™ PCR Master Mix
- NEXTflex™ 16S V5-V6 PCR I Primer Mix
- NEXTflex™ PCR II Barcoded Primer Mix
- Resuspension Buffer
- Nuclease-free Water
Required Materials Not Provided
- 1 ng - 50 ng high-quality genomic DNA in up to 36 µL nuclease-free water for each library
- 96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) or similar
- Adhesive PCR Plate Seal (Bio-Rad®, Cat # MSB1001)
- Agencourt® AMPure® XP 5 mL (Beckman Coulter® Genomics, Cat # A63880)
- Magnetic Stand - 96 (Thermo Fisher Scientific®, Cat # AM10027) or similar
- 2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes
- Nuclease-free barrier pipette tips
- 80% Ethanol, freshly prepared (room temperature)