Universal two-step PCR approach.

Abstract

<p>First-round PCR is performed by using gene-specific primers tailed with a universal linker sequence (light and dark green). The universal tailed amplicons can be used for second-round PCR, where deep sequencing platform-specific adapters can be introduced for either PGM (dark red), Illumina (blue), or 454 (orange). *To prepare PGM amplicon libraries containing trP1 and A adapter sequences on both ends of the library the second-round PCR has to be separated in two reactions with different primer combinations, respectively. All platform-specific amplicon libraries can be prepared with unique barcodes to sequence multiple samples.</p