Introduction to 16S amplicon profiling.

16S rRNA gene sequencing is a widely used method for identifying and comparing bacteria in complex microbial communities. By targeting conserved regions flanking variable regions of the 16S gene, researchers can amplify and sequence short regions that serve as taxonomic fingerprints.

Choosing the right hypervariable region is a critical design decision. Different regions vary in their ability to distinguish closely related organisms, and some primers may amplify certain taxa more efficiently than others. These differences can impact both biological insights and the comparability of your data to other studies.

Modern analysis pipelines typically resolve sequences into amplicon sequence variants (ASVs), error-corrected biological sequences inferred from the reads, down to single-nucleotide resolution. They provide higher resolution than consensus-based clustering (operational taxomonic units) used in the past. For accurate ASV generation, forward and reverse reads must fully span the amplicon and overlap enough to be merged. Region choice affects this overlap, especially when working with shorter reads. Primer selection can also introduce bias by over- or under-representing specific microbial groups.

By aligning your region selection with your scientific question, sample type, and sequencing platform, you can improve taxonomic resolution and reduce technical artifacts.

How region choice affects results

Each 16S region offers trade-offs between resolution, coverage, and technical compatibility. Longer amplicons, such as V1–V3, require longer read lengths (e.g., 2×300 bp) to achieve sufficient overlap for merging. Shorter regions like V4 are well-suited to 2×150 bp or 2×250 bp runs, improving throughput and reducing cost.

V1–V3 often provides strong species-level resolution for skin, oral, and upper respiratory samples but can introduce taxonomic gaps in gut profiles. V3–V4 captures a broad range of taxa with solid genus-level resolution and is widely used in standardized protocols. V4 is favored in high-throughput, longitudinal studies due to its short length and high comparability, though some taxonomic groups may not resolve as well below the genus level.

Primer bias can affect the detection of specific clades. Therefore, aligning your region choice with both your research question and sequencing strategy is essential for reliable results and reproducibility across studies¹.

Revvity offers three region-specific NEXTFLEX™ 16S amplicon-seq kits

1. The NEXTFLEX 16S V1-V3 amplicon-seq kit NEXTFLEX 16S V1-V3 rRNA Amplicon-seq Kits, 12 Barcodes Discover  is ideal for researchers targeting species-level resolution in oral, skin, or respiratory microbiomes, especially with 2×300 bp reads.

   This kit amplifies the V1 through V3 regions of the 16S rRNA gene, generating a relatively long insert (~500 bp). It provides strong species-level resolution for microbial communities where upper-respiratory, skin, or oral taxa are of interest.

   Best-fit applications include nasal and oral microbiomes, where V1–V3 offers improved discrimination among closely related taxa. To ensure successful read merging, this kit is best run with 2×300 bp reads. It is not recommended for platforms or protocols limited to shorter read lengths, as merging rates may drop and reduce ASV recovery.

   Researchers focused on human stool or environments with less representation in the V1–V3 databases may prefer an alternate region for better comparability.

2. The NEXTFLEX 16S V3-V4 amplicon-seq kit NEXTFLEX 16S V3 – V4 Amplicon-Seq Library Prep Kit Discover  balances read length with broad taxonomic coverage and is compatible with 2×250 bp Illumina workflows.

   This kit targets the V3 through V4 regions, generating an amplicon of ~460 bp. It offers broad taxonomic coverage across a wide range of environments and is compatible with 2×250 bp sequencing.

   Use cases include environmental samples, mixed community surveys, and researchers aiming to balance resolution and comparability. The V3–V4 region is frequently used in standardized microbiome studies, offering good genus-level accuracy across body sites and ecosystems.

   Sequencing with 2×250 bp reads typically allows sufficient overlap for merging. For workflows constrained to 2×150 bp, this region may pose challenges for merging and introduce downstream biases in ASV inference.

3. The NEXTFLEX 16S V4 Amplicon-Seq Kit 2.0 NEXTFLEX 16S V4 Amplicon-Seq Kit 2.0 Discover  supports high-throughput studies and is a strong choice for gut microbiome analysis using 2×250 or 2×150 bp reads.

   V4 region spans approximately 250 bp, making it ideally suited for 2×250 bp and 2×150 bp paired-end reads. This compatibility enables cost-effective, high-throughput workflows without sacrificing merging efficiency.

   The kit is well-suited for large-scale longitudinal studies, especially of the gut microbiome, where standardized protocols (e.g., from the Earth Microbiome Project) often use the V4 region. While V4 provides solid genus-level classification, it may underperform in resolving certain closely related species.

   It is a strong choice when throughput, consistency, and cross-study comparability are prioritized. However, if a study requires detailed taxonomic resolution beyond the genus level, V1–V3 or V3–V4 may offer advantages.

Study design and QC that matter regardless of region

Regardless of region choice, good experimental design is critical. Include negative controls and reagent blanks to detect background contaminants. Mock communities with known composition are helpful for benchmarking primer and pipeline performance.

Recommended read depths vary by environment. Soil and ocean samples may require > 50,000 reads per sample, while low-diversity environments like human stool may be adequately profiled with ~ 10,000–20,000 reads. Design batches carefully to minimize run-to-run variability and preserve biological signal.

All NEXTFLEX 16S rRNA amplicon-seq kits are compatible with standard downstream workflows such as DADA2 and Deblur, which infer ASVs after trimming primers and merging reads. Primer-trimming accuracy and consistent preprocessing pipelines help ensure reliable and reproducible results².

Platform and throughput planning

Each kit maps to specific sequencing strategies. The V1–V3 kit is best paired with 2×300 bp reads on Illumina® MiSeq® platforms to ensure full overlap of the longer insert. The V3–V4 kit performs well with 2×250 bp reads, balancing read length with amplicon coverage.

The V4 kit can be run efficiently with 2×150 or 2×250 bp reads, enabling high sample multiplexing per run. Researchers working with limited read budgets or large cohorts often favor V4 for its flexibility and consistent merging performance.

Across all kits, ensure that expected insert length and chosen read length provide sufficient overlap for merging. This is essential for accurate ASV reconstruction and taxonomic assignment.

Use-case driven recommendations

• Human gut longitudinal cohorts: Use the V4 kit for its efficient merging, comparability to published datasets, and compatibility with short-read, high-throughput runs.
• Oral, nasal, or skin studies targeting species-level resolution: Choose V1–V3 if 2×300 bp reads are available. This region offers higher resolution for taxa common to these niches.
• Soil, marine and other environmental microbiome surveys across mixed ecosystems: V3–V4 provides robust coverage and balances taxonomic breadth with established comparability, especially when sequencing with 2×250 bp reads.
• Cross-lab collaborations needing harmonized data: Standardize on the V4 kit when coordinating among collaborators or referencing Earth Microbiome Project protocols. Human gut longitudinal cohorts: Use the V4 kit for its efficient merging, comparability to published datasets, and compatibility with short-read, high-throughput runs.

All three NEXTFLEX 16S amplicon-seq kits generate high-quality libraries when matched to the right region, read length, and study goal. Making an informed region choice early supports consistency, reproducibility, and meaningful biological comparisons.

Quick comparison matrix 

Feature	V1-V3	V3-V4	V4
Typical insert length	~ 500 bp	~ 460 bp	~250 bp
Recommended read length	2x300 bp	2x250 bp	2x150 or 2x250 bp
Beat-fit environments	Oral, skin, nasal	Environmental, mixed sites	Gut, high-throughput cohorts
Throughput suitability	Low-to-mid	Mid	High
Cross-study comparability	Limited	Moderate	High
Resolution/bias notes	High species resolution may miss gut taxa	Balanced, genus-level reliable	Efficient, limited species resolution
When to avoid	No access to 2x300 bp	If limited to 2x150 bp	Species-level profiling required
NEXTFLEX Kitt	NEXTFLEX 16S V1-V3 Amplicon-Seq kit NEXTFLEX 16S V1-V3 rRNA Amplicon-seq Kits, 12 Barcodes Discover	NEXTFLEX 16S V3-V4 Amplicon-Seq kit	NEXTFLEX 16S V4 Amplicon-Seq kit 2.0 NEXTFLEX 16S V4 Amplicon-Seq Kit 2.0 Discover

Reach out to our team if you need support selecting the best kit for your application.