Fig 1.

Microfluidic PCR-based strategies for targeted sequencing. SNPs were detected by combining PCR target enrichment and barcoding with pyrosequencing. (A) The Fluidigm 48.48 Access Array system amplified 48 DNA segments each from 48 sample templates, for a total of 2,304 individual PCRs in a single run. (B) A unique overlapping PCR with two primer pairs that included complementary adapters resulted in sample-specific barcoded amplicons for each amplified gene (48 gene-specific primer pairs were each added to the 48 sample templates together with the 48 sample-specific barcoded primers for the PCR and overlap extension PCR, performed together in a single reaction compartment of the microfluidic system). (C) The 48 amplicons from each sample were collected as 48 sample-specific pools, each with its own barcode. (D) Amplicon pools from the 48 samples were purified, quantitated, and further pooled in an equimolar mixture for subsequent pyrosequencing with the 454 GS FLX sequencer.