How do you prepare your samples for successful amplicon sequencing?

Preparing samples for amplicon-based targeted gene analysis requires specific protocols that differ from other sequencing approaches. This step-by-step guide covers the critical preparation techniques to ensure your samples yield high-quality, bias-free results when using PCR amplification followed by sequencing of specific genomic regions.

Start with pristine DNA

The foundation of any successful amplicon preparation begins with high-quality starting material. For optimal results, extract DNA using methods that minimise fragmentation and contamination. Fresh samples yield better results than archived materials, though properly preserved specimens can still perform well. When extracting DNA specifically for amplicon preparation, avoid harsh mechanical disruption methods that might fragment the DNA below your target amplicon length. Most commercial kits are suitable, but columns that select for high molecular weight DNA often provide better starting material. After extraction, store your DNA in TE buffer or similar stabilising solutions at -20°C with minimal freeze-thaw cycles to preserve integrity.

Master primer design

The success of your experiment hinges on thoughtful primer design. Target-specific primers should flank your region of interest with several critical characteristics:

• Similar melting temperatures between forward and reverse primers (within 2-5°C)
• GC content between 40-60% for consistent amplification
• Minimal secondary structure formation or self-complementarity
• Sufficient specificity to avoid off-target amplification
• Compatible with adding sequencing adapters and barcodes

For microbial studies, consider using primers targeting conserved regions flanking variable segments. For human samples, design primers that avoid common SNPS which could create amplification bias. Design shorter amplicons (under 200bp) to improve success rates when working with degraded samples. Testing primers on control templates before proceeding to valuable samples saves time and resources. Consider running gradient PCR to determine optimal annealing temperatures empirically rather than relying solely on theoretical calculations.

PCR optimisation

The amplification step forms the core of amplicon sequencing and requires careful optimisation. Rather than using standard PCR conditions, take time to optimise for your specific targets.

1. Test multiple polymerase enzymes – high-fidelity versions reduce error rates
2. Determine optimal cycling conditions through empirical testing
3. Find the minimum cycle number that yields sufficient product while reducing bias
4. Validate specificity through gel electrophoresis before proceeding
5. Include appropriate positive and negative controls with every batch

When optimising, focus on minimising chimaera formation, a common artefact in amplicon preparation. This can be achieved by keeping extension times sufficient for complete amplification, minimising cycle numbers, and using polymerases to reduce chimaera formation.

Purification perfection

After amplification, removing primers, nucleotides, and polymerase is essential. Several purification options work well for amplicon preparations:

• Magnetic beads offer size-selective purification that can be calibrated to your amplicon length by adjusting the bead-to-sample ratio. A ratio of 1.8:1 (beads) typically recovers most fragments while removing primers effectively. For more stringent size selection, sequential binding with different ratios can isolate particular size ranges.
• Commercial column-based purification kits provide consistent results but may have lower recovery rates for very short amplicons. Gel extraction offers the most precise size selection but typically results in more significant sample loss and should be reserved for cases where multiple bands are present.

Regardless of method, verify purification success by checking for primer removal using methods like Bioanalyzer or similar fragment analysers that can detect short fragments.

Quantify with precision

Accurate quantification ensures balanced representation when pooling multiple samples. For amplicon libraries, qpcr-based quantification methods that measure only amplifiable DNA provide more reliable results than spectrophotometric methods that measure all DNA regardless of quality.

When working with multiple amplicons of different lengths, remember that molarity (concentration of molecules) rather than mass concentration determines sequencing representation. Calculate molarity based on the concentration and fragment length to ensure proper normalisation before pooling.

The attention to detail during these preparation steps directly translates to higher-quality sequencing data.