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 V4 Amplicon-Seq Kit 2.0 for Illumina® Platforms
Validated library prep for 16S V4 rRNA sequencing using Illumina® sequencing primers
- Fast and easy library prep protocol
- 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 Sciclone NGS and NGSx Workstation to automate your 16S sequencing
- Functionally validated with Illumina® sequencing platforms
The NEXTflex™ 16S V4 Amplicon-Seq Library Prep Kit 2.0 is designed for the preparation of multiplexed amplicon libraries spanning the V4 hypervariable domain of microbial 16S ribosomal RNA (rRNA) genes. These libraries are compatible with paired-end sequencing on the Illumina® MiSeq® sequencing platform and are ideal for monitoring population fluctuations of microbial communities.
Genera measured by analysis of 16S V4 reads obtained from library constructed with the NEXTflex 16S V4 Amplicon-Seq Kit 2.0 from 20 ng of DNA isolated from canine feces.
Optimized Protocol Offers Lower PCR Bias and Fewer Off-target Reads
The NEXTflex 16S V4 Amplicon-Seq Kit 2.0 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 V4 Amplicon-Seq Library Prep Kit 2.0.
384 Barcodes Available for Cost-effective 16S V4 rRNA Sequencing
The NEXTflex 16S V4 Amplicon-Seq Library Prep Kit 2.0 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.
Selected Publications that Reference using the NEXTflex 16S V4 Amplicon-Seq Kit 2.0:
Stalenhoef, J. E., et al. (2017) Fecal Microbiota Transfer for Multidrug-Resistant Gram-Negatives: A Clinical Success Combined With Microbiological Failure. Open Forum Infectious Diseases, 4(2). doi:10.1093/ofid/ofx047.
|NEXTflex™ 16S V4 Amplicon-Seq Kit 2.0 (Barcodes 193 -288)
Automation Next Gen Sequencing
The NEXTflex 16S V4 Amplicon-Seq Kit 2.0 contains enough material to prepare 8, 24, 96 or 192 amplicon-seq libraries from genomic DNA for Illumina® platforms. 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 V4 Amplicon-Seq Kit 2.0 Protocol
- NEXTflex™ PCR Master Mix
- NEXTflex™ 16S V4 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)