Comparison of amplicon-sequencing, pyrosequencing and real-time PCR for detection of YMDD mutants in patients with chronic hepatitis B

Table 1 Results of sequencing, pyrosequencing and real-time PCR for detection of mixed plasmids containing YVDD and YMDD at a final concentration of 10⁶ copies/mL

YVDDplasmidin themixture	Sequencing	Pyrosequencing	Real-timePCR	ΔCt(mean ±  SD)	Calculatedpercentage(mean ± SD)(%)
100%	YVDD	YVDD	YVDD	-0.024 ± 0.06	98 ± 4.0
50%	YVDD	YVDD	YVDD	-0.98 ± 0.07	51 ± 2.5
10%	YMDD	YMDD	YVDD	-3.12 ± 0.09	11 ± 0.7
1%	YMDD	YMDD	YVDD	-6.60 ± 0.14	1 ± 0.1
0.1%	YMDD	YMDD	YVDD	-10.12 ± 0.20	0.09 ± 0.01
0.01%	YMDD	YMDD	YMDD	ND	0

ND: Not detected.

Table 2 Comparison of results obtained by sequencing, pyrosequencing and real-time PCR n (%)

Type	Sequencing	Pyrosequencing	Real-time PCR
rtM204	25 (36)	24 (35)	23 (33)
rtM204I	26 (38)	24 (35)	21 (30)
rtM204V	18 (26)	19 (27)	20 (29)
rtM204I + rtM204V	0 (0)	2 (3)	5 (8)