Summary of currently available targeted long-read sequencing approaches
| Method | DNA input mass per run/prep | Max. number of targets per run | Max. read length | Approx. depth per run/prepa | Platform compatibility | Time |
|---|---|---|---|---|---|---|
| Long-range PCR | ||||||
| ∼10 ng | ∼1000 | ∼10 kb | 10–1000× | ONT, PB | ++ | |
| Notes: Low cost, fast, removes base modifications, methylation marks not preserved | ||||||
| Hybridization methods | ||||||
| 1 µg | ∼20 k | 10 kb | 10–1000× | ONT, PB | +++ | |
| Notes: Probes are expensive, removes base modifications, methylation marks not preserved | ||||||
| CRISPR-Cas9-based approaches | ||||||
| CATCH | 2.75 × 105–1.5 × 106 cells | <10, fewer if targets are of varied sizes | 200 kb–1 Mb | 50–400× | ONT, PB | +++ |
| Notes: In-gel cell lysis and targeting, gives low yield for downstream sequencing, methylation marks preserved | ||||||
| nCATS | 3–10 µg | ≤25, Min. 4 crRNA guides/target, Max. 100 guides/rxn. | 30 kb | 20–850× | ONT | ++ |
| Notes: No background reduction, limited multiplexing options, methylation marks preserved | ||||||
| Single-ended cut | 1–5 µg (10 ng with WGA) | <10, depends on breakpoint/repeat/fusion size | 30 kb | 10–150× | ONT | ++ |
| Notes: Not suited for background reduction or tiling, limited multiplexing options, methylation marks preserved | ||||||
| Negative enrichment/CaBagE | 1–10 µg | ≤25, Min. 4 crRNA guides/target, Max. 100 guides/rxn. | 35 kb | 40–400× | ONT | +++ |
| Notes: Background reduction does not translate to higher coverage, not suited for single-ended cut, limited multiplexing options, methylation marks preserved | ||||||
| ACME | 5–20 µg | ≤25, Min. 4 crRNA guides/target, Max. 100 guides/rxn. | 100 kb | 35–1000× | ONT | ++ |
| Notes: Not suited for single-ended cut, limited multiplexing options, higher input mass required, methylation marks preserved | ||||||
| PureTarget | 2 µg per sample | 20 genes in the predesigned panel | 15 kb | 50–200× | PB | +++ |
| Notes: Limited to predesigned repeat expansion targets, methylation marks preserved | ||||||
| Adaptive sampling | ||||||
| readfish, UNCALLED, ONT built-in, BOSS-RUNS | 1–5 µg | Min. 0.1% Max. 10% of genome size, 1%–5% optimal | Dependent on input DNA size. 4 kb–10 kb | Dependent on target size relative to genome. 10–100× | ONT | + |
| Notes: Requires shearing, not effective on reads <3000 bp, needs many targets, methylation marks preserved | ||||||
[i] (PB) Pacific Biosciences, (ONT) Oxford Nanopore Technologies, (WGA) whole-genome amplification.
[ii] + 1–4 h; ++ 4–8 h; +++ >1 day of preparation.
[iii] aCoverage is highly dependent on the number/length of targets, level of multiplexing, and the type of sequencing run used. These numbers are approximate based on current use cases.