Amplicon Calculators

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An amplicon is the specific DNA fragment produced by PCR amplification between a pair of primers. The amplicon includes the primer sequences and all intervening genomic sequence. Amplicon length is determined by the distance between the primers and affects PCR efficiency, cloning success, and sequencing read coverage. In amplicon sequencing (amplicon-based metagenomics), the same primers are used to amplify a conserved but variable target region (16S rRNA V3–V4, ITS1 for fungi) from all organisms in a mixed sample, generating millions of amplicons that are sequenced to characterize community composition.

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Amplicon Basics

Amplicon = the PCR product defined by a primer pair. Size = forward primer end position − reverse primer end position + 1 (in bases). Typical amplicon lengths by application:

  • qPCR: 80–200 bp (short = efficient amplification, complete extension)
  • Conventional PCR for analysis: 200–1000 bp
  • Long-range PCR: 2–20 kb
  • 16S rRNA amplicons (V3–V4): ~460 bp (Illumina MiSeq compatible)
  • 16S rRNA (V4 region only): ~254 bp (shorter, higher throughput)

16S rRNA Amplicon Sequencing

The 16S rRNA gene contains 9 hypervariable regions (V1–V9) flanked by conserved sequences used as universal primer binding sites. Amplifying V3–V4 or V4 with Illumina paired-end sequencing enables identification of bacteria to genus/species level. Workflow: extract DNA from sample → PCR amplify target region → add Illumina adapters and sample barcodes → pool and sequence on MiSeq → DADA2 or QIIME2 bioinformatics pipeline → ASV/OTU table.

Amplicon Quality Control

Critical QC: check amplicon size and purity on agarose gel (single band expected); use NTC (no-template control) to detect contamination; verify amplification efficiency from standard curve; use high-fidelity polymerase to minimize PCR errors; check for primer dimers (appear at ~100 bp on gel or bioanalyzer).

Glossary

Amplicon
The DNA fragment produced between two PCR primers; includes primer sequences and intervening template; size determined by primer positions; used in diagnostics, cloning, and metagenomics.
16S rRNA Amplicon Sequencing
Culture-independent bacterial community profiling using universal primers targeting hypervariable regions of the 16S rRNA gene; analyzes millions of amplicons to identify community members.
ASV (Amplicon Sequence Variant)
An exact denoised sequence from amplicon sequencing (DADA2/Deblur); single-nucleotide resolution; reproducible across studies; replaces OTUs in modern microbiome analysis.

Frequently Asked Questions

An amplicon is the DNA fragment produced between two PCR primers during amplification — it includes the primer sequences and all intervening template sequence. Amplicon size = distance from forward primer to reverse primer (inclusive). Amplicon specificity depends on primer design and annealing temperature. In diagnostics, the amplicon sequence is verified by Sanger sequencing. In amplicon-based metagenomics, billions of amplicons from the same target region across all organisms in a sample are sequenced simultaneously to characterize community composition.

16S rRNA amplicon sequencing uses universal primers targeting conserved regions flanking variable sequences (V1–V9) within the bacterial 16S rRNA gene. Most commonly, V3–V4 (~460 bp) or V4 alone (~254 bp) is amplified from extracted community DNA. After Illumina sequencing, reads are processed by pipelines (QIIME2, DADA2) to produce: denoised amplicon sequence variants (ASVs) or clustered OTUs; taxonomic assignments by comparison to reference databases (Silva, Greengenes2, NCBI); and diversity metrics (alpha, beta diversity). It enables culture-independent characterization of microbiomes in gut, soil, water, and clinical samples.

For qPCR, amplicons should be 80–200 bp: short amplicons (80–120 bp) give the most efficient, consistent amplification; complete extension in the extension step (Taq extends ~1000 bp/min at 72°C, so 30 s easily extends 500 bp). Long amplicons (>300 bp in qPCR) reduce efficiency and increase Ct variability. Short amplicons also improve efficiency when RNA quality is low (degraded mRNA is fragmented — primers spanning exon-exon junctions with short amplicons can still amplify). For amplicons spanning exon-exon junctions, verify no genomic DNA contamination using no-reverse-transcriptase controls.

OTU (Operational Taxonomic Unit): sequences clustered at 97% identity threshold — traditionally the proxy for bacterial species. OTU approaches bin diverse sequences together, potentially hiding true diversity within the 3% variation window. ASV (Amplicon Sequence Variant): exact sequence after denoising using DADA2 or Deblur — single-nucleotide resolution without arbitrary clustering. ASVs are reproducible across studies (exact sequences can be compared across datasets); OTUs are study-specific. Modern microbiome studies prefer ASVs because they are more precise and comparable. QIIME2 generates ASVs by default using the DADA2 plugin.