TABLE 2.
Confirmation of the designed universal EV cloning method efficiency in generating infectious clones
| Virus name | Length (nucleotides) | EV species | Colonies picked/total observed colonies | Positive colonies confirmed by colony PCR/total picked coloniesa (%)b | NGS analysisc |
Rescue confirmation (DNA/RNA) | |
|---|---|---|---|---|---|---|---|
| No. of determined reads | No. of determined reads mapping against the wild type (%) | ||||||
| EV-A71 | 7,400 | A | 10/41 | 8/10 (80) | 135,842 | 135,842 (100) | +/+ |
| CV-B5 | 7,402 | B | 10/45 | 8/10 (60) | 186,241 | 186,241 (100) | +/+ |
| ECHO6 | 7,418 | B | 10/38 | 6/10 (70) | 125,654 | 125,654 (100) | +/+ |
| ECHO30 | 7,428 | B | 10/36 | 7/10 (70) | 92,165 | 92,165 (100) | +/+ |
| CV-A24 | 7,458 | C | 10/64 | 5/10 (50) | 112,267 | 112,267 (100) | +/+ |
| EV-D68 | 7,333 | D | 10/45 | 8/10 (80) | 86,253 | 86,253 (100) | +/+ |
a
From the picked positive colonies, one sample of each EV serotype was subjected to further analysis using deep sequencing and rescue confirmation.
b
Cloning efficiency was calculated based on the method described by Beaty et al. (26).
c
Deep sequencing was performed using Illumina’s Nextera XT DNA library preparation kit. Determined reads were demultiplexed, quality trimmed, and filtered using CLC Genomics Workbench 7.0 at a minimum variant read frequency of 2%.