Reliable multiplex sequencing with rare index mis-assignment on DNB-based NGS platform

Li, Qiaoling; Zhao, Xia; Zhang, Wenwei; Wang, Lin; Wang, Jingjing; Xu, Dongyang; Mei, Zhiying; Liu, Qiang; Du, Shiyi; Li, Zhanqing; Liang, Xinming; Wang, Xiaman; Wei, Hanmin; Liu, Pengjuan; Zou, Jing; Shen, Hanjie; Chen, Ao; Drmanac, Snezana; Liu, Jia Sophie; Li, Li; Jiang, Hui; Zhang, Yongwei; Wang, Jian; Yang, Huanming; Xu, Xun; Drmanac, Radoje; Jiang, Yuan

doi:10.1186/s12864-019-5569-5

BMC Genomics

Table 2 Level of contamination for PCR-free library on BGISEQ-500

From: Reliable multiplex sequencing with rare index mis-assignment on DNB-based NGS platform

a. Sample arrangement of PCR-free library (HPV)
Template	YH-1	*HPV11 + YH*	YH-1	YH-1	YH-1	YH-1	H2O-1	YH-1	YH-1	YH-1	YH-1	YH-1	Barcode 1
Sample index	MGIP-1	MGIP-2	MGIP-3	MGIP-4	MGIP-5	MGIP-6	MGIP-7	MGIP-8	MGIP-9	MGIP-10	MGIP-11	MGIP-12	Barcode 1
Template	YH-2	YH-2	H2O-2	YH-2	YH-2	YH-2	YH-2	YH-2	YH-2	*HPV18 + YH*	YH-2	YH-2	Barcode 2
Sample index	MGIP-13	MGIP-14	MGIP-15	MGIP-16	MGIP-17	MGIP-18	MGIP-19	MGIP-20	MGIP-21	MGIP-22	MGIP-23	MGIP-24	Barcode 2
Template	YH-3	YH-3	YH-3	YH-3	*HPV31 + YH*	YH-3	YH-3	YH-3	YH-3	YH-3	YH-3	YH-3	Barcode 3
Sample index	MGIP-25	MGIP-26	MGIP-27	MGIP-97	MGIP-29	MGIP-30	MGIP-31	MGIP-32	MGIP-33	MGIP-34	MGIP-35	MGIP-36	Barcode 3
Template	YH-4	YH-4	YH-4	YH-4	YH-4	YH-4	*HPV33 + YH*	YH-4	YH-4	YH-4	YH-4	YH-4	Barcode 4
Sample index	MGIP-37	MGIP-38	MGIP-39	MGIP-40	MGIP-41	MGIP-42	MGIP-43	MGIP-44	MGIP-45	MGIP-46	MGIP-47	MGIP-48	Barcode 4
Template	*HPV52 + YH*	YH-5	YH-5	YH-5	YH-5	H2O-5	YH-5	YH-5	YH-5	YH-5	YH-5	YH-5	Barcode 5
Sample index	MGIP-49	MGIP-50	MGIP-51	MGIP-52	MGIP-53	MGIP-54	MGIP-55	MGIP-56	MGIP-57	MGIP-58	MGIP-59	MGIP-60	Barcode 5
Template	YH-6	YH-6	YH-6	YH-6	YH-6	YH-6	YH-6	H2O-6	*HPV45 + 11 + YH*	YH-6	YH-6	YH-6	Barcode 6
Sample index	MGIP-61	MGIP-62	MGIP-63	MGIP-64	MGIP-65	MGIP-66	MGIP-67	MGIP-68	MGIP-69	MGIP-70	MGIP-71	MGIP-72	Barcode 6
b. Performance of SeqHPV
Library Index	Sample Index	Total Reads	Mapped Reads	Mapped Rate	Major Types	HBB Score (0-10)	HPV Score (0-10)	False Positive	False Negative
1	MGIP002	2470768	1800287	72.90%	HPV11,HBB	10	10	0	0
2	MGIP022	2653747	2526477	95.20%	HPV18,HBB	10	10	0	0
3	MGIP029	1793620	690665	94.30%	HPV31,HBB	10	10	0	0
4	MGIP043	1511740	1210189	80.10%	HPV33,HBB	10	10	0	0
5	MGIP049	1641545	1447782	88.20%	HPV52,HBB	10	10	0	0
6	MGIP069	2800830	1942883	69.40%	HPV45,HPV11,HBB	10	10	0	0
8	MGIP002	8	4	50.00%	HPV11,HBB	5	10	0	0
	MGIP029	4	3	75.00%	HPV31	0	10
	MGIP049	17	16	94.10%	HPV52	0	10
	MGIP069	11	7	63.60%	HPV45,HBB	10	10
c. Index contamination rate of PCR-free libraries
	Library index	HBB	HPV11	HPV18	HPV31	HPV33	HPV52	HPV45
Read depth	1	2994608	*1348826*	83	36	14	23	33
	2	2722311	75	*2310955*	31	17	24	31
	3	1891540	53	65	*1566954*	10	8	15
	4	2936888	54	90	80	*940365*	18	25
	5	2289158	61	52	14	14	*1237126*	22
	6	1747934	*253390*	53	17	9	18	*1497716*
	8	27	3	0	3	0	16	5
Percent of read depth	1		*14.7309%*	0.0009%	0.0004%	0.0002%	0.0003%	0.0004%
	2		0.0008%	*25.2386%*	0.0003%	0.0002%	0.0003%	0.0003%
	3		0.0006%	0.0007%	*17.1132%*	0.0001%	0.0001%	0.0002%
	4		0.0006%	0.0010%	0.0009%	*10.2700%*	0.0002%	0.0003%
	5		0.0007%	0.0006%	0.0003%	0.0002%	*13.5110%*	0.0002%
	6		*2.7673%*	0.0006%	0.0002%	0.0001%	0.0002%	*16.3570%*
	8		0.0000%	0.0000%	0.0000%	0.0000%	0.0002%	0.0001%

a. Positive samples are in italic bold, negative samples with YH genome only are in black font, water controls are bolded and sample index are in italic. b. Empty controls are in italic. Index 7 data was excluded due to its oligo synthesis contamination. c. Italic bold, proper combinations; italic, improper combinations. The average sample-to-sample mis-assignment rate is 0.0004% without any filtering

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