NEXTflex™ 16S V4 Amplicon-Seq Library Prep Kit

Validated amplicon-seq library prep for bacterial metagenomics studies

Catalog# Product Name Quantity US List Price
4201-01 NEXTflex™ 16S V4 Amplicon-Seq Kit (with 4 barcodes) 16 rxns $360 Buy Now
4201-02 NEXTflex™ 16S V4 Amplicon-Seq Kit (with 12 barcodes) 48 rxns $844 Buy Now
4201-03 NEXTflex™ 16S V4 Amplicon-Seq Kit (with 24 barcodes) 96 rxns $1,604 Buy Now
4201-04 NEXTflex™ 16S V4 Amplicon-Seq Kit (with 48 barcodes) 192 rxns $3,048 Buy Now
4201-05 NEXTflex™ 16S V4 Amplicon-Seq Kit (with 96 barcodes) 384 rxns $5,790 Buy Now
4201-07 NEXTflex™ 16S V4 Amplicon-Seq Kit (with 288 barcodes) 1,152 rxns $14,913 Buy Now

NEXTflex Illumina 16S V4 Amplicon-Seq Kit
  • Fast - 2 hour library prep kit
  • Easy - Two step protocol
  • Low input - Only 50 ng of input required
  • Maximized recovery rates
  • Flexible barcode options– Kits containing 4, 12, 24, 48, 96 or 288 unique barcodes
  • Sequencing primers are included in the kit
  • Automation-friendly workflow is compatible with liquid handlers
  • Functionally validated with Illumina sequencing platforms

The NEXTflex™ 16S V4 Amplicon-Seq Kit is a validated amplicon-seq library prep kit developed to simplify bacterial metagenomics studies using Illumina® HiSeq and MiSeq platforms.

Fast, Two-Step Library Prep Protocol

The NEXTflex™ 16S V4 Amplicon-Seq Kit offers a fast two step, gel-free protocol that reduces library prep time to 2 hours and allows for complete automation of bacterial metagenomics studies. There are two main steps involved in the NEXTflex 16S V4 Amplicon-Seq Kit protocol: amplification and cleanup. Primers included in the NEXTflex 16S V4 Amplicon-Seq Kit are designed to hybridize to the fourth hypervariable (V4) domain of microbial 16S ribosomal RNA (rRNA) genes, allowing for amplification and sequencing of this variable region. The single cleanup step ensures maximum recovery of amplicons for downstream sequencing.

Users interested in different degrees of multiplexing should contact

The NEXTflex 16S V4 Amplicon-Seq Kit utilizes an excellent phylogenetic marker, allowing for studies of entire microbial communities or individual taxa, and is ideal for monitoring population fluctuations.

Bioo Scientific's NEXTflex™ 16S V1-V3 Amplicon-Seq Kit has recently been launched to enhance metagenomics studies.

Selected Publications that Reference using the NEXTflex 16S V4 Amplicon-Seq Kit:

Dalmassoa, A., et al. (2016) Characterization of microbiota in Plaisentif cheese by high-throughput sequencing. LWT - Food Science and Technology. 69, 490–496. doi:10.1016/j.lwt.2016.02.004.

Davitt, J. L., (2014) "An Assessment of Broiler Litter Bacterial Diversity using Next Generation DNA Sequencing." Master's Thesis, University of Tennessee,

Dees, M. W., Lysøe, E., Nordskog, B. and Brurberg, M. B. (2015) Bacterial Communities Associated with Surfaces of Leafy Greens: Shift in Composition and Decrease in Richness over Time. Appl. Envir. Microbiol., 81: 1530 - 1539. doi: 10.1128/AEM.03470-14.

Franchini P, Fruciano C, Frickey T, Jones JC, Meyer A (2014) The Gut Microbial Community of Midas Cichlid Fish in Repeatedly Evolved Limnetic-Benthic Species Pairs. PLoS ONE 9(4): e95027. doi:10.1371/journal.pone.0095027.

Liu, S., et al. (2016) The Host Shapes the Gut Microbiota via Fecal MicroRNA. Cell Host & Microbe. 19:1, 32–43. doi: 10.1016/j.chom.2015.12.005.

Lukens, J. R. et. al. (2014) Dietary modulation of the microbiome affects autoinflammatory disease. Nature. doi:10.1038/nature13788.

Lyons, P. P., Turnbull, J. F., Dawson, K. A. and Crumlish, M. (2016) Effects of low-level dietary microalgae supplementation on the distal intestinal microbiome of farmed rainbow trout Oncorhynchus mykiss (Walbaum). Aquaculture Research. doi: 10.1111/are.13080.

Lyons, P. P., Turnbull, J. F., Dawson, K. A. and Crumlish, M. (2015) Exploring the microbial diversity of the distal intestinal lumen and mucosa of farmed rainbow trout Oncorhynchus mykiss (Walbaum) using next generation sequencing (NGS). Aquaculture Research. doi: 10.1111/are.12863.

Man, S. M. et al. (2015) Critical Role for the DNA Sensor AIM2 in Stem Cell Proliferation and Cancer. Cell. doi:10.1016/j.cell.2015.06.001.

Martelli, F. S., et al. (2016) Long-term efficacy of microbiology-driven periodontal laser-assisted therapy.  Eur J Clin Microbiol Infect Dis. 35:423–431. doi: 10.1007/s10096-015-2555-y.

Zeller, G. et al. (2014) Potential of fecal microbiota for early-stage detection of colorectal cancer. Mol Syst Biol. 10:766. doi:10.15252/msb.20145645.

Kit Specs

The NEXTflex 16S V4 Amplicon-Seq Kit contains enough material to prepare 16, 48, 96, 192, 384, or 1,152 samples of bacterial genomic DNA for Illumina® compatible sequencing. 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 Protocol


 NEXTflex 16S V4 Amplicon-Seq Kit Protocol

Kit Contents

NEXTflex™ DNA PCR Master Mix
NEXTflex™ 16S V4 Forward Primer (5 µM)
NEXTflex™ 16S V4 Reverse Primer Barcode (5 µM) 1 – 4
Resuspension Buffer
Nuclease-free Water
Custom Sequencing Read Primer 1 (100 µM)
Custom Sequencing Read Primer 2 (100 µM)
Custom Index Read Primer (100 µM)

Required Materials not Provided

50 ng high-quality genomic DNA in up to 34 µL nuclease-free water.
96 well PCR Plate Non-skirted (Phenix Research, Cat # MPS-499) / or / similar 
96 well Library Storage and Pooling Plate (Fisher Scientific, Cat # AB-0765) / or / similar
Adhesive PCR Plate Seal (BioRad, Cat # MSB1001)
Agencourt AMPure XP 5 mL (Beckman Coulter Genomics, Cat # A63880)
Magnetic Stand -96 (Ambion, Cat # AM10027) / or / similar
2, 10, 20, 200 and 1000 µL pipettes / multichannel pipettes
Nuclease-free barrier pipette tips
1.5 mL nuclease-free microcentrifuge tubes
80% Ethanol, freshly prepared (room temperature)


How many samples can be multiplexed using the NEXTflex 16S V4 Amplicon-Seq Kit?

Currently barcodes are available for multiplexing up to 288 samples together in a single Illumina flow cell. The exact number of barcodes that can be used will be based on experimental design.


On which sequencing platforms can libraries prepared using the NEXTflex 16S V4 Amplicon-Seq Kit be run?

The 16S V4 libraries can be sequenced on any Illumina platform including the MiSeq, NextSeq 500 and the HiSeq 2500.


What are the input requirements for the NEXTflex 16S V4 Amplicon-Seq Kit?

Only 50 ng of DNA is required for library construction.


Does the bacterial DNA need to be isolated from the host DNA prior to library preparation?

No, because this kit uses primers which hybridize upstream and downstream of the fourth hypervariable (V4) domain of microbial 16S ribosomal RNA (rRNA) genes, DNA from non-bacterial sources will not be amplified and does not need to be removed prior to library preparation.


Does the NEXTflex 16S V4 Amplicon-Seq library preparation kit contain sequencing primers?

Yes, the kit does include the sequencing primers needed for Illumina sequencing.


How long is this library preparation protocol?

This kit utilizes a two step library preparation protocol that only requires 2 hours to complete.


Can this kit be used to sequence variable regions of the 16S rRNA gene other then the V4 region?

No; however, we will be launching the NEXTflex 16S V1-3 Amplicon-Seq Kit in the near future.


Are custom kits available that allow for multiplexing a different number of samples than are listed on the webpage?

The NEXTflex 16S V4 Amplicon-Seq Kit Kits contain 4, 12, 24, 48, 96 or 288 unique barcodes for multiplexing samples. If you want to multiplex a different number of samples, contact Bioo Scientific at to discuss your multiplexing needs and we should be able to help you.

Sequences of NEXTflex 16S V4 Amplicon-Seq Reverse Primers - Excel / PDF

Instructions for installing NEXTflex Barcode Indices in Illumina Experiment Manager