Table 1.
Observed frequency of the CRISPR/Cas9 adenosine insertion, specific to the GE rice line in rice seed samples (n°1–11). The samples were composed of DNA extracted from rice seeds of the GE rice line and its parental rice line. The samples contained 100%, 99.9%, 99.1%, 95%, 90%, 50%, 10%, 5%, 0.9%, 0.1%, or 0% of the GE rice line. For each sample, the copy number associated with the GE rice line, previously measured using a 2-plex ddPCR method [16], is indicated (Table S1). The targeted high-throughput sequencing approach was performed in quadruplicate for each sample, allowing us to subsequently determine the allele frequency of the CRISPR/Cas9 adenosine insertion specific to the GE rice line. The associated depth of coverage of the reference at the position of this specific indel (bp 679,646) is indicated.
| Rice Seed Sample Description | CRISPR/Cas9 Adenosine Insertion | ||||||
|---|---|---|---|---|---|---|---|
| Sample n° | GE Rice Line Content | Allele Frequency ~ | Depth of Coverage ~ | ||||
| Percentage | Copy Number | Detection | Percentage | StDev | Value | StDev | |
| 1 | 100 | 13,733.3 | + | 99.92 | 0.03 | 119,592 | 29,499 |
| 2 | 99.9 | 13,346.7 | + | 99.86 | 0.03 | 97,038 | 25,007 |
| 3 | 99.1 | 13,273.3 | + | 99.73 | 0.02 | 70,873 | 12,339 |
| 4 | 95 | 12,113.3 | + | 97.97 | 0.15 | 101,581 | 63,687 |
| 5 | 90 | 10,753.3 | + | 92.06 | 0.24 | 75,787 | 36,739 |
| 6 | 50 | 5640.0 | + | 53.80 | 0.57 | 71,791 | 19,099 |
| 7 | 10 | 1153.3 | + | 8.01 | 0.29 | 86,841 | 10,531 |
| 8 | 5 | 528.0 | + | 4.00 | 0.15 | 98,283 | 40,588 |
| 9 | 0.9 | 85.3 | + | 0.66 | 0.01 | 101,464 | 33,345 |
| 10 | 0.1 | 10.8 | + * | 0.13 * | 0.02 | 126,724 | 42,631 |
| 11 | 0 | 0 | − | 0.00 | 0.00 | 158,436 | 99,002 |
~ For each sample, the average value of the 4 replicates and the associated standard deviation (StDev) are indicated. * The CRISPR/Cas9 adenosine insertion was observed in 3 of the 4 replicates.