Table 2.

Chikungunya alternating passages unique mutations

Feature (gene)	Genome position	Reference allele	Read coverage	Variant allele	Amino acid position	Amino acid change	Variant rate
nsp4	6001	C	233 902	A	112	N > K	0.62075
E1	10380	A	116 688	C	129	synonymous	0.18479
nsp4	5950	C	112 016	U	95	synonymous	0.07434
nsp4	6843	A	82 162	G	393	D > G	0.02060
nsp3	4919	A	172 536	C	282	synonymous	0.01508
E2	9701	G	294 074	C	387	G > A	0.01431
nsp4	5910	G	95 074	U	82	R > I	0.01418
nsp2	2234	U	77 920	C	185	F > L	0.01384
6K	9967	A	179 958	U	53	S > C	0.01082

Using our pipeline, we were able to collect all the positions carrying any variant allele with a frequency >0.1% from every sequenced sample (the starting virus population (passage 1), eight cycles in Hela cells and alternating passages between Hela cells and mosquito C6 cells) and pinpoint unique positions across the viral genome in which variants were found only in the alternating passages positions. One of these variant positions, found in the viral polymerase protein (nsp4) demonstrated a high variant allele rate which may suggest functional relevance for survival in alternating host passages.