Description: Guide to Amplicon Sequencing

The Guide to Amplicon Sequencing provides a complete overview of how to prepare, design, and validate amplicon libraries for high-throughput DNA sequencing. This document explains every step of the workflow from sample preparation and primer design to PCR amplification, purification, and quality assessment using the Agilent BioAnalyzer.

It introduces the use of Fusion Primers, which combine a target-specific 3′ region with system-specific 5′ tags (Primer A and Primer B) optimized for the Genome Sequencer 20 System. These primers enable accurate, clonal amplification through emulsion PCR (emPCR), allowing single-molecule sequencing without bacterial cloning.

The guide details:

• Critical DNA quality and quantity requirements for reliable amplification.
• Reagents, instruments, and materials needed for amplicon library preparation.
• Step-by-step PCR setup and purification protocols using SPRI beads.
• Guidelines for quantification via PicoGreen fluorometry and fragment analysis.
• Practical examples of fusion primer design and optimized thermocycling programs.
• Troubleshooting tips for removing primer dimers and ensuring single, high-quality amplicon peaks.

This technical manual is an essential reference for molecular biologists and sequencing specialists aiming to perform accurate targeted DNA sequencing, mutation detection, or genetic marker analysis using amplicon-based workflows.