Fig. 1

Experimental design. (a) Design of single and dual-index sequencing strategy and schematic describing the 3 amplicon designs: Fusion Primer Design (A) is a one step PCR which uses a single 12-nt error-correcting Golay index sequence (blue) allowing a high multiplexing capability. Tag tailed design (B) is a 2-step PCR which uses a universal primer for the first step and a dual index barcoded primer set in the second step. Standard Illumina Nextera 8-nt index sequences were used (pink Index 5; blue Index 7). The Pac Bio Ligate Adapters design (C): Two harpin adapters (grey) were ligated to a barcoded template (BF forward barcode; BR reverse barcode) to allow multiplexing. (b) Platform Specific Amplicon Libraries: Illumina paired-end sequencing (1,2) generates 2 sequencing reads (R1 and R2) per each cluster and can have single (Standard/Golay) or dual indexes (I5, I7). Ion Torrent and 454 (3) have a single read for each bead with a single index (MID). Pacific Bioscience generate a single circular read for each molecule (SMRT bell) and can have one (BF or BR) or two indexes. The starting point and direction of sequencing reads are indicated by a solid blue line and arrows, respectively. In the case of Fusion Primer Design custom sequencing primer were used