Full-lengthmsp2was amplified in a first round of PCR that used primers annealing to the conserved 5' and 3' ends of themsp2gene. In the nested PCR that followed,msp2amplicons were asymmetrically barcoded using primers from the conserved 5' and 3' regions of the gene but annealing directly downstream and upstream of the annealing sites for the forward and reverse oligos, respectively, used in the first PCR reaction. Asymmetric amplicon barcoding allowed for the accurate pooling and computational identification of up to 384 individual samples using a combination of 40 different barcodes. Thereafter, SMRTbell adaptors were coupled to the barcoded amplicons, resulting in single-stranded circularized constructs containing both strands ofmsp2. Circularizedmsp2amplicons were sequenced in 32 polymerase passes (16 on each strand), and the resulting sequences for each amplicon were aligned to produce circular consensus reads.
