TABLE 5.

Optimization of mlrPCR-sWGA and whole-genome amplification reactions

Variable	EBV input copies for sWGAa	Denaturation buffer	Avg EBV input (genome copy/μl)	Avg human input (β-actin/μl)	dNTP composition (G/C/T/A ratio)	Whole-genome amplification incubation time (h), temp (°C)	EBV DNA output (copy/ng)	Human DNA output (β-actin/ng)	EBV enrichmentb	Human enrichmentc	EBV enrichment/human enrichment
Input EBV copies, dNTP composition	10,000	TE	62,448	3,456	30/30/10/10	16 h, 30°C	4,573	7	1.06	0.03	35.33
	1,000	TE	10,038	13,911	30/30/10/10	16 h, 30°C	505	59	1.01	0.08	12.63
	100	TE	467	10,883	30/30/10/10	16 h, 30°C	55	70	2.28	0.12	19.00
	10	TE	10	11,326	30/30/10/10	16 h, 30°C	4	68	8.26	0.12	68.83
	1	TE	6	19,483	30/30/10/10	16 h, 30°C	0	55	0.21	0.06	3.50
	0	TE		11,551	30/30/10/10	16 h, 30°C		76		0.23	0.00
	10,000	TE	62,448	3,456	30/30/5/5	16 h, 30°C	4,847	2	1.12	0.01	112.00
	1,000	TE	10,038	13,911	30/30/5/5	16 h, 30°C	544	75	1.09	0.11	9.91
	100	TE	467	10,883	30/30/5/5	16 h, 30°C	72	65	3	0.12	25.00
	10	TE	10	11,326	30/30/5/5	16 h, 30°C	3	65	5.97	0.11	54.27
	1	TE	6	19,483	30/30/5/5	16 h, 30°C	0	81	0.22	0.09	2.44
	0	TE		11,551	30/30/5/5	16 h, 30°C		71		0.21	0.00
	10,000	TE	62,448	3,456	15/15/5/5	16 h, 30°C	3,483	16	0.81	0.07	11.57
	1,000	TE	10,038	13,911	15/15/5/5	16 h, 30°C	488	84	0.97	0.12	8.08
	100	TE	467	10,883	15/15/5/5	16 h, 30°C	73	109	3.04	0.19	16.00
	10	TE	10	11,326	15/15/5/5	16 h, 30°C	1	80	2.71	0.14	19.36
	1	TE	6	19,483	15/15/5/5	16 h, 30°C	0	103	0.25	0.11	2.27
	0	TE		11,551	15/15/5/5	16 h, 30°C		118		0.35	0.00
	10,000	TE	62,448	3,456	15/15/2/2	16 h, 30°C	3,324	12	0.77	0.05	15.40
	1,000	TE	10,038	13,911	15/15/2/2	16 h, 30°C	533	77	1.06	0.11	9.64
	100	TE	467	10,883	15/15/2/2	16 h, 30°C	78	105	3.24	0.19	17.05
	10	TE	10	11,326	15/15/2/2	16 h, 30°C	4	105	7.8	0.18	43.33
	1	TE	6	19,483	15/15/2/2	16 h, 30°C	0	110	0	0.12	0.00
	0	TE		11,551	15/15/2/2	16 h, 30°C		108		0.32	0.00
Denaturation buffer, incubation time, and temperature	10	TE	10	11,326	30/30/5/5	8 h, 30°C	2	40	6.31	0.14	45.07
	10	TE	10	11,326	30/30/5/5	16 h, 30°C	2	40	8.74	0.14	62.43
	10	TE	10	11,326	30/30/5/5	8 h, 35°C	1	22	4.05	0.08	50.63
	10	TE	10	11,326	30/30/5/5	16 h, 35°C	2	22	6.14	0.08	76.75
	10	TE+Q sol	10	11,326	30/30/5/5	8 h, 30°C	2	41	9.44	0.14	67.43
	10	TE+Q sol	10	11,326	30/30/5/5	16 h, 30°C	3	60	13.94	0.21	66.38
	10	TE+Q sol	10	11,326	30/30/5/5	8 h, 35°C	3	28	10.72	0.1	107.20
	10	TE+Q sol	10	11,326	30/30/5/5	16 h, 35°C	4	49	15.13	0.17	89.00

^aTotal EBV genomes = 20 μl of input sWGA.

^bCalculated as “(EBV_post/EBV_pre)/(DNA_post/DNA_pre).”

^cCalculated as “(human_post/human_pre)/(DNA_post/DNA_pre).”