Figure 2

Primer and fragment design for library preparation. The P. falciparum gene fragments of interest are amplified with gene-specific primers, in a “gene-specific PCR” (1). The primers anneal to loci in the P. falciparum genome, while encoding non-annealing overhangs, which are then incorporated into the PCR products during amplification. The non-annealing overhangs are identical on all gene-specific primers, and serve as annealing site for the index primers (2), which can in turn anneal to all amplified gene fragments. The index primers also encode individual 8-base indices as well as an adaptor sequence. The index and adapter sequence are incorporated into the PCR product (denoted amplicon from here on), which can then bind to the Miseq flow cell through the adaptor sequence, and be identified through sequencing of it’s unique index combination.