Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing

Table 4 Read coverage and mutations in seven genes identified by using indexed amplicon sequencing

Gene locus*	Amplicon size (bp)	Consensus length (bp)	Total read counts for 96 DNA pools	Read coverage per sample			Base changes	Type of base change					Amino acid substitutions	Misssense mutations	Nonsense mutations	Total number of base changes expected per M2' plant**	Distance between base changes (kb)***
Gene locus*	Amplicon size (bp)	Consensus length (bp)	Total read counts for 96 DNA pools	Average	Minimum	Maximum	Base changes	G > A	C > T	T > A	A > T	Others	Amino acid substitutions	Misssense mutations	Nonsense mutations	Total number of base changes expected per M2' plant**	Distance between base changes (kb)***
Glyma06g19820	7539	7519	3476540	54	13	137	88	46	30	1	4	7	30	29	1	7224	132
Glyma05g01770	5643	5625	3844251	80	5	172	67	26	35	1	2	3	26	25	1	7348	129
Glyma08g46520	2531	2514	1476507	69	18	166	77	31	36	2	6	2	44	43	1	18828	50
Glyma06g23026	1304	1294	4156094	381	33	1188	32	17	12	1	0	2	12	10	2	15188	63
Glyma20g22160	6390	6370	6977332	130	35	276	132	61	56	1	3	11	72	69	3	12785	74
Glyma11g15580	4214	4190	1155194	32	8	124	101	36	35	0	3	27	28	27	1	14833	64
Glyma20g25000	2648	2623	1044416	47	10	137	64	26	26	2	9	1	21	21	0	14958	64
Total	30269	30135	22130334				561						233	224	9	(av.) 14269	(av.) 67.0
Percentage of each type of base change****								43.3 %	41.0 %	1.4 %	4.8 %	9.4 %

*Gene locus names are indicated according to gene models in the Glyma_189 assembly (v1.1)
**The total number of base changes per M2' plant was calculated from a total number of base changes in the library estimated from the amplicon size and the size of chromosome-scale assembly of the soybean genome (950,068,807 bp) and then divided by the total number of plants (1536)
***The distance between base changes was calculated from the size of the amplicon divided by the total number of base changes found in the library
****The percentage of each type of base change was calculated from a total each type of base change in seven genes divided by all base changes

ISSN: 1471-2164