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Two-step PCR strategy adopted for high throughput sequencing. On the first-step PCR the specific gene section (target) is amplified with a forward primer (blue) bearing a universal tag (red), and a reverse primer (blue) carrying a trP1 adapter (green). For the second-step PCR the amplicons of the first step are amplified with a reverse primer that matches the trP1 adapter (green) and a forward bar-coded (pink) primer (brown) for each desired pool of individuals and genes.
