NEXTFLEX 16S V3 – V4 Amplicon-Seq Library Prep Kit

Simple 16S V3–V4 workflow, beads included, flexible multiplexing

The kit uses a two-PCR design. PCR I generates the V3–V4 amplicon from genomic DNA, then a bead cleanup removes primers and small fragments. PCR II adds the barcoded PCR II primers for indexing, followed by a final cleanup and pooling.

NEXTFLEX NGS Cleanup Beads are supplied with the kit. This helps reduce variability and avoids separate consumables purchase.

Indexing scales with your study: choose 48 or 96 barcode sets, with expanded options available up to 384 unique PCR II barcodes (upon request). Each barcode includes two reactions.

Accurate V3–V4 taxonomic assignment with overlapping reads and flexible input

Paired-end 2×300 sequencing spans the ~610 bp V3–V4 library and yields overlapping forward and reverse reads. That overlap enables read merging, quality-aware error modeling, and chimera filtering, which improves separation of closely related taxa and reduces misclassification in downstream calls.

Figure 1: Population results displayed as the percentage of total 16S DNA present after sequencing the extracted Zymo Community Standard

Accurate assignment matters because it shapes the conclusions you report, including community composition, relative abundance, and changes across conditions. Using exact sequence variants instead of broad clusters improves resolution and comparability across studies, which is especially helpful for longitudinal designs, method validation, and meta-analysis.

The kit supports a practical input of 1–50 ng DNA, so typical extracts can go straight into PCR without extra concentration. To keep results consistent across that range, the protocol provides cycle-number guidance matched to input, which helps achieve sufficient yield while limiting over-amplification artifacts.

Not sure when to choose 16S or metagenomics for microbial work? Read a short comparison that outlines the tradeoffs.

Cosmos-Hub 16S analysis (included with “S” variants)

Product variants above with “S” in their SKU number include access to Cosmos-Hub, an online analysis platform that converts your 16S V3–V4 FASTQ files into interpretable results using a standardized, version-controlled workflow. You receive a QC-filtered ASV table with taxonomy, clear quality summaries, and publication-ready visualizations. Comparative analysis tools are built in, including tables, heatmaps, bar and stacked plots, multiple alpha and beta diversity metrics, abundance distribution views, differential abundance testing, and statistics between groups. Results export as tab-separated tables and PDFs, and each chart can be downloaded as PNG or SVG for downstream use . Learn more about Cosmos-Hub.

Revvity 16S rRNA analysis - online demonstration

Now you can schedule a one-on-one 30-minute online demonstration that will cover everything you need to know about the Cosmos-Hub® which is now available with Revvity’s NEXTFLEX™ 16S rRNA Amplicon-seq Kits. You will be able to see the analysis demonstrated and our experts will be able to answer any questions that you may have.

Automation-ready

Automation protocols are available for the Sciclone™ NGS and NGSx workstations, and the Sciclone NGSx iQ™ workstation supporting walkaway NGS library preparation and higher throughput in 96-well plates while reducing manual steps and variability.

Region options to fit your study

Revvity offers 16S library prep kits for V3–V4, V4, and V1–V3, so you can match the target region to your goals: choose V3–V4 when you want more phylogenetic signal across two adjacent hypervariable domains, pick V4 for the shortest and most widely standardized amplicon that fits common read lengths, or select V1–V3 when your assay or consortium prefers that region for higher precision in certain clades or to align with established SOPs.