Lib a

Each amplicon was purified from agarose gel by QIAquick Gel Extraction kit (Qiagen) and then by Agencourt AMPure XP beads (Beckman Coulter) using 1.6:1 ratio of beads to sample. Products were quantified by dsDNA HS Qubit® Assay Kit (Life Technologies), fourteen samples were pooled in equivalent amounts and of 3 × 10⁷ DNA copies were subjected to emulsion PCR using GS Junior Titanium emPCR Kit (Lib-A). After initial denaturation at 94°C for 1 minute, the reaction was run for 50 cycles of 94°C for 30 seconds, 58°C for 4 minutes and 30 seconds, and 68°C for 30 seconds. DNA library beads enrichment was carried out according to the emPCR Amplification Method Manual Lib-A (Roche), with the exception that the number of bead washes was 15. The required input of 500 000 enriched beads was loaded onto the Pico Titer Plate (PTP) and sequencing was carried out for 200 cycles using full processing mode for amplicons (GS Junior Sequencer, 454/Roche). In total, two independent pyrosequencing runs were performed (14 samples with specific MID were pooled in each).