Fig. 5

Mutant discovery by using HRM and indexed amplicon sequencing. DNA extracted from M2’ plants was preserved as the original DNA stock in 96-well plates. The DNA pool in a 384-well plate (four samples per pool) was used for both methods. After a mutation was detected by HRM analysis, base changes in four original DNA samples were confirmed by direct sequencing. If the mutation was found to be silent, HRM analysis and direct sequencing of other regions were performed. In indexed amplicon sequencing, 7 target gene regions (1.3–7.5 kb, 30.3 kb in total) were amplified by long-range PCR. The amplicons of four samples were further pooled. The 96 samples were indexed by using a transposome-based Nextera XT Index kit. Bulk read data for all 96 DNA pools were obtained from Miseq and mapped onto the reference sequences of target genes after classification of the DNA pool by using indices. Base changes at high frequency in many reads were treated as a mutation and were filtered by using a Glyma_189 gene annotation to exclude mutations that did not lead to amino acid substitutions. Based on the information from DNA pool classification with indices, the base change and the plant in which it occurred could be determined by direct sequencing of each of the 16 original M2’ DNA samples. Amplicon sequencing using NGS allows rapid and effective detection of DNA pools containing mutations that cause desirable functional amino acid substitutions