Fig. 4

Scheme of next generation-based high-throughput multiplex amplicon sequencing and a representative result. a Target regions amplified by nested PCR. In the second PCR, primers with tags for the third PCR were used. In the third PCR, the primers had unique pairs of indices for multiplex sequencing. After sequencing with MiSeq, the reads obtained were separated according to the indices. Red and blue lines represent index sequences with priming targets for Illumina sequencing. b Snapshot of the mapped reads for sample ID A1 with IGV. Arrowheads represent the called variants. The reference is the sequence for the 3D7 strain. c Validation by Sanger sequencing of the corresponding region. Arrowheads represent polymorphisms detected by sequencing