Table 3.

Comparison of wild type and mutant frequencies of P. falciparum in mosquito samples using Sanger sequencing and NGS

Gene	Sanger sequencing of mosquito samples			NGS of mosquito samplesa		Sequenced samples by both methods	p-value
	Wild type %	Mutant %	Mixture %	Wild type %	Mutant %
K13
Y493H R539T I543T C580Y	100	0	0	100	0	66	1
pfcrt
CVMNK (72–76)	100	0	0	100	0	93	1
pfmdr1 F1
N86Y/F	98.8	0	1.2	100	0	81	3.44 × 10−4
Y184F	46.9	21.0	32.1	50.3	49.3		0.59
pfmdr1 F2
S1034C N1042D D1246Y	100	0	0	100	0	70	1
pfdhps F1
S436F/A	100	0	0	100	0	86	1
A437G	2.3	95.3	2.3	0	100		1.32 × 10−7
pfdhps F2
K540E	2.3	96.6	1.1	6.2	93.7	87	0.17
A581G A613T/S	100	0	0	100	0		1
pfdhfr
C50R/S	99	1.0	0	100	0	95	1.64 × 10−4
N51I	0	100	0	0.9	98.4		1
C59R	3.2	32.6	64.2	3.6	96.3		1
S108N	0	100	0	0.3	96.3		1
164L	100	0	0	100	0		1

Fisher’s exact test was utilized for all comparisons except for pfmdr1 position Y184F, for which χ-squared test was performed. Sanger sequencing mixtures were assumed as mutant for statistical calculations. F1: fragment 1. F2: fragment 2. Wild-type haplotypes are indicated on the left and mutant amino acids on the right of the position number (see Additional file 1: Tables S5 and S7)

^aNGS allele frequency estimates were calculated by dividing individual unfiltered allele depth by total filtered depth, hence total values do not always add up to 100 (see “Methods” section) [61]