Microarray for Hepatitis B Virus Genotyping and Detection of 994 Mutations along the Genome

DOI: 10.1128/JCM.00344-10

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FIG. 1.
Concordance between sequencing and the microarray for genotyping according to viral load.

TABLE 1.

Numbers of analyzed positions and polymorphisms

ORF^a	Coded protein or promoter	No. of analyzed codons		No. of probed polymorphisms
ORF^a	Coded protein or promoter	Previous version	Improved version	Previous version	Improved version
P	Polymerase terminal protein (tp)	2	0	2 (deletions)	0
	Polymerase spacer (sp)	2	2	10	10
	Polymerase reverse transcriptase (rt)	26	66	70	197
S	Envelope (PreS1)	1	3	4	6
	Envelope (PreS2)	2	6	6	16
	Envelope (S)	54	98	150 (+3 deletions)	339
	Basal core promoter (BCP nucleotides)	15	33	38 (+16 deletions and insertions)	112
	PreCore (preC)	12	14	35 (+4 deletions)	55
	Core (C)	11	21	27 (+1 insertion and 1 deletion)	115
X	X	18	55	81 (+1 deletion)	144
Total		143	298	433	994

TABLE 2.

Genotyping performance

Genotype (sequencing)	No. of samples	Mean Id score	Highest/second-highest Id score ratio	Sensitivity (%)^a	Specificity (%)^b
A	54	89.3	1.2	87	99.3
B	17	72.1	1.24	94.1	100
C	15	70.2	1.17	93.3	96
D	54	70.3	1.26	96.3	98.5
E	23	61.5	1.23	100	99.4
F	12	55.2	1.26	100	100
G	1	70.2	1.63	100	99.5
H	7	55.2	1.28	100	100
A+C	3	84.6^c	1.13	66.7	100
A+G	4	48.9^d	1.25	100	100

↵ a Sensitivity was calculated as the percentage of samples referenced as a given genotype by sequencing that were identified as the same genotype by the array.
↵ b Specificity was calculated as the percentage of samples identified as a given genotype by the array and a different genotype by sequencing.
↵ c Detected as A or C.
↵ d Detected as A or G.

TABLE 3.

Detection of wild-type and mutant mixtures by the microarray

Ratio of rt180/rt204mt vs rt180/rt204wt	Amino acid identified at a total plasmid concentration (copies/ml) of:^a
Ratio of rt180/rt204mt vs rt180/rt204wt	1 × 10³		1 × 10⁴		1 × 10⁶
0	rt180L	rt204M	rt180L	rt204M	rt180L	rt204M
0.1	rt180L	rt204M	rt180L	rt204M	rt180L	rt204M
0.2	rt180L	rt204V	rt180L	rt204V	rt180L	rt204V
0.3	rt180L	rt204V	rt180L	rt204V	rt180L	rt204V
0.4	rt180L	rt204V	rt180L	rt204V	rt180L	rt204V
0.5	rt180M	rt204V	rt180L	rt204V	rt180L	rt204V
0.6	rt180M	rt204V	rt180L	rt204V	rt180M	rt204V
0.7	rt180M	rt204V	rt180M	rt204V	rt180M	rt204V
0.8	rt180M	rt204V	rt180M	rt204V	rt180M	rt204V
0.9	rt180M	rt204V	rt180M	rt204V	rt180M	rt204V
1	rt180M	rt204V	rt180M	rt204V	rt180M	rt204V

↵ a Two plasmids with the whole HBV genome were constructed: rt180/rt204wt, with leucine and methionine at positions rt180 and rt204, respectively, and rt180/rt204mt, with methionine and valine at positions rt180 and rt204, respectively. Mixtures of the two plasmids were assembled at different total plasmid concentrations and ratios of rt180/rt204mt versus rt180/rt204wt. The italicized letters indicate the amino acids that were identified by the microarray.

TABLE 4.

Comparison of sequencing and microarray for detection of amino acids at 16 selected positions^a

Position	No. of samples	Microarray identification			No. of samples identified by sequencing as:
		Microarray identification					WT																					MUT					Ind
				Type	Amino acid	I	V	L	T	A	M	N	P	W	G	S	T	L	G	M	P	V	S	A	N	I	Y	R	D	H	C	*	Ind
rt169	164	WT	I	161													1
		MUT	T												0
			L													0	1
		Ind															1
rt173	164	WT	V		150												1														1
		MUT	L		3											8															1
			G														0
		Ind																													0
rt180	164	WT	L			104																									1
		MUT	M			2												28
			C																0
		Ind				14^b												13^c													2
rt181	164	WT	A				124																								1
		MUT	T														2
			G																	0
			V				2										12
		Ind					20^d								2^d		1^d	0
rt184	164	WT	T				152																	1
		MUT	S				1														1										2
			A																			0
			G				1										0
			N																				0
			I																					1
		Ind					2																	3
rt194	164	WT	A					164
		MUT	T												0
		Ind																													0
rt202	163	WT	S											155
		MUT	I																					0
			G											5			0
		Ind												3^e																	0
rt204	164	WT	M						104											1				2							3
		MUT	I						1															10
			V						2											21				12
			S																		0
		Ind							3^f											3^f				1^f							0
rt236	123	WT	N							57
		MUT	T												2
			Y																						0
		Ind								58					6																0
rt250	114	WT	M						107
		MUT	V						5											0
		Ind							1^g																						1^h
s120	163	WT	P								53																				3
		MUT	T								1								0
			G														104
			A														2					0
		Ind																													0
s145	162	WT	G										56
		MUT	R										1													0
			A																			1
			T												1				10		2
			P																70							1
			S																1		13
		Ind																													0
preC28	29	WT	W									17																		2
		MUT	*									2																		8
			R																							0
		Ind																													0
preC29	22	WT	G										22
		MUT	D																								0
			H																									0
			S																		0
		Ind																													0
BCP1762	39	WT	A					14							2																1
		MUT	T					2							10
			G					2							4		0
		Ind						2							2																0
BCP1764	39	WT	G										14									1
		MUT	T										1		1							4							1
			A										2									8
			C										1									1							0
		Ind																				5									0

↵ a Samples with a reference Sanger sequence were tested with the microarray. The numbers of samples with a reference differ according to the codon and are indicated for each position. For each codon, either a wild type or one of the possible mutants (MUT) was identified by sequencing or the microarray. Ind, indeterminate; *, stop codon.
↵ b Eleven arginines, 2 phenylalanines, 1 proline.
↵ c Twelve arginines, 1 lysine.
↵ d Twenty-three prolines.
↵ e One valine, 1 cysteine and 1 glutamic acid.
↵ f Seven glycine.
↵ g Alanine.
↵ h Isoleucine by sequencing and threonine with the microarray.