Accurate taxonomic assignment with full 16S V4 overlap and flexible input

Paired-end 2×300 sequencing fully covers the V4 insert, so forward and reverse reads overlap across every base. That overlap enables pair-merging, model-based error correction, and chimera removal, which improves discrimination among closely related bacteria and reduces misclassification in downstream taxonomic calls.

Figure 1: Genus-level composition from 16S V4 libraries prepared with the NEXTFLEX 16S V4 Amplicon-Seq Kit 2.0. 

Accurate assignment matters because it drives the conclusions you report: community composition, relative abundance, and changes across conditions. Using exact sequence variants rather than broad clusters increases resolution and comparability across studies, which is especially useful for longitudinal work, method validation, and meta-analysis.

The kit supports a practical input range of 1–50 ng genomic DNA, so typical extractions can go straight into PCR without additional concentration. To maintain consistency across that range, the protocol provides cycle-number guidance that is adjusted to input amount, which helps achieve sufficient yield while limiting over-amplification artifacts.

Not sure when to choose 16S over metagenomics for microbial research? Read a quick 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 moves your 16S V4 FASTQ files to interpretable results with a standardized, version-controlled workflow, enabling fast and easy analysis of complex microbiome data. You receive a quality-controlled ASV table with taxonomy, clear QC summaries, and publication-ready visualizations. Comparative analysis is built in, including tables, heatmaps, bar and stacked plots, multiple alpha and beta diversity indices, abundance distribution views, differential abundance testing, and statistics between groups. Exports are provided as tab-separated tables and PDFs, with options to download individual charts as PNG or SVG files 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. Please choose a timeframe that suits you best and we will provide you a Microsoft Teams personal link to join when the time comes. We look forward to speaking with you.

Automation-ready

Automation protocols are available for the Sciclone NGS and NGSx workstations, supporting walkaway NGS library preparation and higher throughput in 96-well plates while reducing manual steps and variability. The Sciclone G3 NGSx iQ workstation adds an on-deck thermal cycler to automate PCR steps end to end. Together, these options standardize results and streamline batching without changing the kit chemistry.

Choose the 16S region that fits your study

Revvity offers 16S library prep kits for V4, V3–V4, and V1–V3 so you can align region choice with your objectives: use V4 when you want the shortest and most widely adopted amplicon that fits common read lengths and large legacy datasets, choose V3–V4 when added phylogenetic signal across two adjacent hypervariable domains will help resolution, or select V1–V3 when your assay or consortium specifies that region or certain clades are best distinguished there.

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