Amplicon sequencing 101

Amplicon sequencing — also called targeted sequencing — focuses on sequencing specific regions of the genome rather than the whole thing. This lets researchers concentrate on genes or regions of interest, cutting cost and complexity while increasing depth and accuracy in those regions.

Primer panel vs. tiling scheme

Both produce a set of primer pairs, but they answer different questions.

Primer panel

• Designed for a specific list of regions (typically gene exons).
• Variable coverage — only the regions you cared about.
• Clinical/research use: cancer hotspot panels, pharmacogenomics, inherited-disease screening.

Tiling scheme

• Overlapping amplicons across a whole reference sequence.
• Complete coverage with no gaps (e.g. a 30 kb viral genome covered by 75 amplicons in 2 pools).
• Use case: full-genome sequencing of viruses, plasmids, or small contigs.

Why pools matter

Adjacent amplicons share overlapping primer-binding regions. If two adjacent primer pairs were in the same multiplex PCR they'd compete and produce primer dimers. Splitting amplicons into pools — each pool run as a separate reaction — avoids that. Two pools is the usual default; more pools improve robustness for tightly overlapping designs.

What “quality” means for a primer

• Tm: melting temperature, kept in a tight band (59.5–62.5 °C by default) so all primers anneal at the same cycle temperature.
• GC content: 40–65 % to balance binding stability with specificity.
• Hairpin ΔG: secondary structures lower than ~47 °C are tolerated.
• Homopolymer runs: capped at ~5 bp to avoid slippage.
• GC clamp: helps polymerase priming at the 3′ end.