Next-generation sequencing traces human induced pluripotent stem cell lines clonally generated from heterogeneous cancer tissue

Table 1 Read number (original)

	Original
Sample	No. of reads	Read length (b)	No. of bases (Gb)
NCC1	18260718	101	3.7
	18260718	101
CC1	16706190	101	3.4
	16706190	101
CC1-1	13045740	101	2.6
	13045740	101
CC1-2	17725772	101	3.6
	17725772	101
CC1-7	14780507	101	3.0
	14780507	101
CC1-8	17311972	101	3.5
	17311972	101
CC1-9	16664067	101	3.4
	16664067	101
CC1-11	15455638	101	3.1
	15455638	101
CC1-12	15391361	101	3.1
	15391361	101
CC1-17	19009957	101	3.8
	19009957	101
CC1-18	19746313	101	4.0
	19746313	101
CC1-25	15492560	101	3.1
	15492560	101

NCC1: The matched adjacent non-cancerous tissue; CC1: The starting cancer tissue of the induced pluripotent stem cell lines; CC1-1 to CC1-25: Each induced pluripotent stem cell line.

Table 2 Read number (modified)

	Modified1
Sample	No. of reads	Read length (b)	Ratio (%) (Mod/Ori)
NCC1	18146940	1012	99.38
	18146940	1012	99.38
CC1	16597436	1012	99.35
	16597436	1012	99.35
CC1-1	12942132	1012	99.21
	12942132	1012	99.21
CC1-2	17614866	1012	99.37
	17614866	1012	99.37
CC1-7	14687008	1012	99.37
	14687008	1012	99.37
CC1-8	17180329	1012	99.24
	17180329	1012	99.24
CC1-9	16545785	1012	99.29
	16545785	1012	99.29
CC1-11	15346749	1012	99.30
	15346749	1012	99.30
CC1-12	15281269	1012	99.28
	15281269	1012	99.28
CC1-17	18880292	1012	99.32
	18880292	1012	99.32
CC1-18	19618808	1012	99.35
	19618808	1012	99.35
CC1-25	15378555	1012	99.26
	15378555	1012	99.26

¹Modified read file is a data set from the original read file with the adapter sequences and low-quality bases removed;

²Therefore, there were reads shorter than the number indicated by the read length (b) in a portion of the modified read file. NCC1: The matched adjacent non-cancerous tissue; CC1: The starting cancer tissue of the induced pluripotent stem cell lines; CC1-1 to CC1-25: Each induced pluripotent stem cell line.

Table 3 Mapped reads

	NCC1	CC1	CC1-1	CC1-2	CC1-7	CC1-8	CC1-9	CC1-11	CC1-12	CC1-17	CC1-18	CC1-25
No. of total reads ①	36293880	33194872	25884264	35229732	29374016	34360658	33091570	30693498	30562538	37760584	39237616	30757110
No. of mapped reads ② (③+ ④ + ⑤)	36210841	33066194	25545096	34845596	28911555	33842665	32976780	29898986	30464308	36670526	38078508	30173511
No. of mapped reads with Paired-End ③	26935180	26333748	21884450	25981140	21498822	26807330	26704280	25386830	25103580	29513694	30869186	21622036
No. of mapped reads with Single-End ④	15741	24050	14051	16508	12336	17984	14651	27429	14354	27336	25456	19687
No. of discarded reads1 ⑤	9259920	6708396	3646595	8847948	7400397	7017351	6257849	4484727	5346374	7129496	7183866	8531788
No. of unmapped reads (①‐②)	83039	128678	339168	384136	462461	517993	114790	794512	98230	1090058	1159108	583599
No. of effective reads (③ + ④)	26950921	26357798	21898501	25997648	21511158	26825314	26718931	25414259	25117934	29541030	30894642	21641723

¹Discarded reads were as follows: Reads mapped to chromosomes other than the target; Reads where each paired-end is mapped to a different chromosome; Reads not used for single-nucleotide variant/InDel detection such as PCR duplicates. Each number of ② consists of each total number of "③ plus ④ plus ⑤"; "① - ②" means "each number of ① minus each number of ②"; "③ + ④" means "each number of ③ plus each number of ④". NCC1: The matched adjacent non-cancerous tissue; CC1: The starting cancer tissue of the iPSC lines; CC1-1 to CC1-25: Each induced pluripotent stem cell line.

Table 4 Sequence depth

	Theoretical depth1		Observed depth2
Sample	Total bases (Mb)	Depth3	Effective bases on target (Mb)	Average depth on target
NCC1	3689	1173.31	1399	445.06
CC1	3375	1073.43	1084	344.89
CC1-1	2635	838.23	999	317.89
CC1-2	3581	1138.94	1279	406.72
CC1-7	2986	949.69	1195	380.13
CC1-8	3497	1112.35	1352	430.01
CC1-9	3366	1070.72	1355	431.06
CC1-11	3122	993.07	1077	342.50
CC1-12	3109	988.94	1359	432.12
CC1-17	3840	1221.45	1407	447.66
CC1-18	3989	1268.76	1559	495.99
CC1-25	3129	995.45	1083	344.37

¹Theoretical depth calculated from the total number of bases obtained by DNA sequencing;

²Observed depth used for single-nucleotide variant/InDel identification;

³Theoretical depth [Total Bases (Mb)]/[Target region (Mb)]. Target region: SureSelect Human Kinome Kit (approximately 3.1 Mb). NCC1: The matched adjacent non-cancerous tissue; CC1: The starting cancer tissue of the induced pluripotent stem cell lines; CC1-1 to CC1-25: Each induced pluripotent stem cell line.

Table 5 Target capture

	NCC1	CC1	CC1-1	CC1-2	CC1-7	CC1-8	CC1-9	CC1-11	CC1-12	CC1-17	CC1-18	CC1-25
Initial bases on target ①	3143812	3143812	3143812	3143812	3143812	3143812	3143812	3143812	3143812	3143812	3143812	3143812
Initial bases near target ②	3790645	3790645	3790645	3790645	3790645	3790645	3790645	3790645	3790645	3790645	3790645	3790645
Initial bases on or near target ③	6934457	6934457	6934457	6934457	6934457	6934457	6934457	6934457	6934457	6934457	6934457	6934457
Total effective reads ④	26950921	26357798	21898501	25997648	21511158	26825314	26718931	25414259	25117934	29541030	30894642	21641723
Total effective bases (Mb) ⑤	2663	2602	2157	2573	2125	2643	2637	2503	2480	2914	3047	2133
Read length mean (b)	98.91	98.83	98.58	99.02	98.84	98.61	98.77	98.58	98.79	98.74	98.69	98.65
Read length median (b)	101	101	101	101	101	101	101	101	101	101	101	101
Effective bases on target (Mb) ⑥	1399	1084	999	1279	1195	1352	1355	1077	1359	1407	1559	1083
Effective bases near target (Mb) ⑦	440	342	355	405	380	438	448	327	445	430	450	342
Effective bases on or near target (Mb) ⑧	1839	1426	1354	1683	1575	1790	1804	1403	1804	1837	2009	1425
Fraction of effective bases on target (%) (⑥/⑤)	52.54	41.67	46.32	49.70	56.25	51.14	51.38	43.01	54.78	48.30	51.18	50.75
Fraction of effective bases near target (%) (⑦/⑤)	16.52	13.14	16.45	15.73	17.88	16.56	17.00	13.05	17.96	14.75	14.77	16.05
Fraction of effective bases on or near target (%) (⑧/⑤)	69.06	54.82	62.78	65.43	74.12	67.70	68.38	56.06	72.74	63.05	65.95	66.80
Average sequencing depth on target (⑥/①)	445.06	344.89	317.89	406.72	380.13	430.01	431.06	342.50	432.12	447.66	495.99	344.37
Average sequencing depth near target (⑦/②)	116.05	90.22	93.63	106.76	100.21	115.48	118.31	86.16	117.51	113.43	118.68	90.33
Average sequencing depth on or near target (⑧/③)	265.21	205.68	195.30	242.75	227.11	258.07	260.10	202.38	260.14	264.96	289.74	205.50
Base covered on target ⑨	3143221	3143152	3142784	3143540	3143280	3143263	3143277	3142887	3143338	3143035	3143296	3142818
Coverage of target region (%) (⑨/①)	99.98	99.98	99.97	99.99	99.98	99.98	99.98	99.97	99.98	99.98	99.98	99.97
Base covered near target ⑩	3775671	3773869	3774076	3771915	3768892	3773823	3776942	3760218	3776060	3766215	3762031	3762823
Coverage of near target region (%) (⑩/②)	99.60	99.56	99.56	99.51	99.43	99.56	99.64	99.20	99.62	99.36	99.25	99.27
Fraction of target covered with at least 15 × (%)	99.72	99.62	99.59	99.69	99.65	99.68	99.70	99.55	99.69	99.60	99.68	99.58
Fraction of target covered with at least 8 × (%)	99.86	99.78	99.78	99.83	99.81	99.82	99.83	99.76	99.83	99.78	99.83	99.77
Fraction of target covered with at least 4 × (%)	99.93	99.89	99.89	99.90	99.90	99.90	99.91	99.87	99.92	99.89	99.91	99.89
Fraction of flanking region covered with at least 15 × (%)	86.88	84.24	87.52	85.84	84.62	87.92	89.85	79.85	90.17	84.50	80.72	82.36
Fraction of flanking region covered with at least 8 × (%)	94.32	93.13	94.66	93.66	92.95	94.70	95.77	89.93	95.87	92.55	89.96	91.39
Fraction of flanking region covered with at least 4 × (%)	97.85	97.33	97.91	97.50	97.15	97.87	98.27	95.76	98.32	96.86	95.71	96.37

The target region, as covered by the SureSelect Human Kinome Kit, was approx. 3.1 Mb. Near target region was 200 bases forward and backward of the target region. "⑥/⑤" means "each number of ⑥ devided by each number of ⑤"; "⑦/⑤" means "each number of ⑦ devided by each number of ⑤"; "⑧/⑤" means "each number of ⑧ devided by each number of ⑤"; "⑥/①" means "each number of ⑥ devided by each number of ①"; "⑦/②" means "each number of ⑦ devided by each number of ②"; "⑧/③" means "each number of ⑧ devided by each number of ③"; "⑨/①" means "each number of ⑨ devided by each number of ①"; "⑩/②" means "each number of ⑩ devided by each number of ②". NCC1: The matched adjacent non-cancerous tissue; CC1: The starting cancer tissue of the induced pluripotent stem cell lines; CC1-1 to CC1-25: Each induced pluripotent stem cell line.

Table 6 Chromosome number, genome position, reference vs single-nucleotide variant, novelty vs dbSNP135, gene symbol, and mutation types of single-nucleotide variants

SNV No.	Chromo-some No.	Genome position	Ref.|SNV	Novel/known	Gene symbol	Mutation types
1	chr2	37336419	C|T	Novel	EIF2AK2	Missense
2	chr2	179408086	A|G	Novel	TTN	Missense
3	chr3	41705179	A|C	Novel	ULK4	Missense
4	chr5	112769527	C|T	Novel	TSSK1B	Missense
5	chr5	180048626	C|T	Novel	FLT4	Missense
6	chr6	31947203	T|C	Novel	STK19	Missense
7	chr7	23808650	G|T	Novel	STK31	Missense
8	chr7	98490141	G|C	Novel	TRRAP	Missense
9	chr12	1009680	C|T	Novel	WNK1	Missense
10	chr16	23690401	C|T	Novel	PLK1	Missense
11	chr17	8789811	G|A	Novel	PIK3R5	Nonsense
12	chr17	37881392	A|G	Novel	ERBB2	Missense
13	chr19	2046399	G|A	Novel	MKNK2	Missense

Ref.: The allele of the human reference genome hg19; SNV: Single-nucleotide variant.

Table 7 Allelic depth of single-nucleotide variants among the matched adjacent non-cancerous tissue, the starting cancer tissue, and the cancer tissue-derived induced pluripotent stem cell lines

	Allelic depth of SNVs
SNV No.	NCC1	CC1	CC1-1	CC1-2	CC1-7	CC1-8	CC1-9	CC1-11	CC1-12	CC1-17	CC1-18	CC1-25
1	250|0	246|0	232|0	250|0	250|0	250|0	250|0	248|0	250|0	250|0	129|121	250|0
2	249|0	240|0	240|0	248|0	248|1	250|0	129|121	248|0	242|0	250|0	250|0	244|0
3	246|0	247|2	249|0	238|1	246|0	248|0	233|0	241|0	238|1	241|0	245|0	132|106
4	250|0	239|0	243|0	248|0	245|0	120|129	250|0	236|0	250|0	250|0	250|0	249|0
5	216|0	150|0	75|79	189|0	184|0	180|0	200|1	131|0	176|0	221|0	207|0	179|0
6	249|0	238|0	250|0	132|114	250|0	250|0	242|0	248|0	248|0	250|0	250|0	249|0
7	250|0	248|0	250|0	250|0	245|0	246|0	245|0	135|111	249|0	250|0	250|0	246|1
8	233|0	240|1	243|0	250|0	245|0	242|0	247|0	248|0	132|113	240|1	247|0	241|0
9	249|0	246|2	250|0	250|0	249|0	220|30	244|0	249|0	250|0	249|1	250|0	249|0
10	247|0	177|0	188|0	119|121	198|0	244|0	241|0	176|0	221|0	224|0	249|0	174|0
11	246|1	172|0	181|0	208|0	209|0	198|0	189|0	175|0	244|0	182|0	233|0	95|87
12	249|1	195|54	241|0	249|0	249|0	249|1	249|0	250|0	249|0	250|0	249|1	250|0
13	137|0	91|10	79|0	131|0	102|0	103|0	103|0	83|0	106|0	111|0	142|0	90|0

NCC1: The matched adjacent non-cancerous tissue; CC1: The starting cancer tissue of the induced pluripotent stem cell lines; CC1-1 to CC1-25: Each induced pluripotent stem cell line; SNV: Single-nucleotide variant.

Table 8 Genotypes of single-nucleotide variants among the matched adjacent non-cancerous tissue, the starting cancer tissue, and the cancer tissue-derived induced pluripotent stem cell lines

	Genotypes of SNVs
SNV No.	NCC1	CC1	CC1-1	CC1-2	CC1-7	CC1-8	CC1-9	CC1-11	CC1-12	CC1-17	CC1-18	CC1-25
1	C/C	C/C	C/C	C/C	C/C	C/C	C/C	C/C	C/C	C/C	C/T	C/C
2	A/A	A/A	A/A	A/A	A/A	A/A	A/G	A/A	A/A	A/A	A/A	A/A
3	A/A	A/A	A/A	A/A	A/A	A/A	A/A	A/A	A/A	A/A	A/A	A/C
4	C/C	C/C	C/C	C/C	C/C	C/T	C/C	C/C	C/C	C/C	C/C	C/C
5	C/C	C/C	C/T	C/C	C/C	C/C	C/C	C/C	C/C	C/C	C/C	C/C
6	T/T	T/T	T/T	T/C	T/T	T/T	T/T	T/T	T/T	T/T	T/T	T/T
7	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/T	G/G	G/G	G/G	G/G
8	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/C	G/G	G/G	G/G
9	C/C	C/C	C/C	C/C	C/C	C/T	C/C	C/C	C/C	C/C	C/C	C/C
10	C/C	C/C	C/C	C/T	C/C	C/C	C/C	C/C	C/C	C/C	C/C	C/C
11	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/A
12	A/A	A/G	A/A	A/A	A/A	A/A	A/A	A/A	A/A	A/A	A/A	A/A
13	G/G	G/A	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/G	G/G

NCC1: The matched adjacent non-cancerous tissue; CC1: The starting cancer tissue of the induced pluripotent stem cell lines; CC1-1 to CC1-25: Each induced pluripotent stem cell line; SNV: Single-nucleotide variant.