Compilation of UL library preparation protocols, optimized in human GM12878 cells
| Previous protocols | New protocols | ||||||||
|---|---|---|---|---|---|---|---|---|---|
| Quick's UL, Quick 2018 | Logsdon's UL, Logsdon 2020 | Rocky Mountain Tyson 2019 | Krazy StarFish | ULK001 | RAD004-Nemo | ULK001-Nemo | ULK114 | ULK114-Nemo | |
| A | B | C | D | E | F | G | H | I | |
| Input cell numbera | 50 million | 20–70 million | >2 million | >2 million | 6 million | 1–6 million | 1–6 million | 6 million | 6 million |
| Input DNA amount | 15 µg | 2–3 µg | >7.5 µg | >7.5 µg | 40 µg | 5–40 µg | 5–40 µg | 40 µg | 40 µg |
| ONT library preparation kit | RAD004 | RAD004 | LSK109 | RAD004 | ULK001 | RAD004 | ULK001 | ULK114 | ULK114 |
| Fragmentation volume | Small (15 µL) | Small (18 µL) | Medium (60–100 µL) | Medium (2 × 100 µL) | Large (1 mL) | Large (0.5–1 mL) | Large (0.5–1 mL) | Large (1 mL) | Large (1–1.2 mL) |
| Library cleanup protocol | No | No | No | Partial using filter paper | Nanobind (circulomics) | Nemo | Nemo | No | Nemo |
| Processing time (excluding incubations) | 15 min | 2.5 h | 18–24 h | 30 min | ∼2.5 h | ∼2.5 h | ∼2.5 h | ∼2.5 h | ∼2.5 h |
| Input library per MinION flow cell | 10–15 µg | 2–3 µg | 1–3 µg | 2–4 µg | 5–6 µg | 1–5 µg | 1–5 µg | NA | NA |
| No. of loads per library (MinION) | 1 | 1 | 1–3 | 3 | 6 | 1–6 | 1–6 | NA | NA |
| Expected yield per MinION flow cell | ∼3 Gb | 1–2 Gb | 10–>20 Gb (depends on N50) | 6–8 Gb (3 loads) | >20 Gb (3 loads) | >15 Gb (3 loads) | ∼20 Gb (3 loads) | NA | NA |
| Input library per PromethION flow cell | NA | NA | NA | NA | 10–13 µg | 10–13 µg | 10–13 µg | 10–13 µg | 10–13 µg |
| No. of loads per library (PromethION) | NA | NA | NA | NA | 3 | 1–3 | 1–3 | 3 | 3–4 |
| Expected yield per PromethION flow cell (Gb) | NA | NA | NA | NA | NA | NA | 24 Gb (1 load) | ∼85 Gb | up to >100 Gb |
| UL read length (>100 kb) | Yes | Yes | No | Yes | Yes | Yes | Yes | Yes | Yes |
[i] (NA) Not available.
[ii] aCell number was dependent on the extraction protocol used; here, it is listed as the number of cells required by the original protocol. If cell number is unknown, use the DNA amount.