Table 1.
Summary of long-read DNA enrichment methods
Method | Typical read length | Typical coverage | Example of percent reads on-target | Example of number of targets | Platform | Advantages | Disadvantages |
---|---|---|---|---|---|---|---|
PCR | 7 kb | 50–1,000× | >96%141 | 19 amplicons31 | ONT, PacBio |
Simple design High enrichment Low hands-on time Easy to multiplex samples Easy to multiplex targets Sensitive Low input |
Erases native DNA modifications Limited fragment length Lengthy optimization may be required Introduction of PCR errors Multiple reactions often needed |
Hybridization capture | 5 kb | 200–1,000× | 66.3%42 | 4,800 genes34 | ONT, PacBio |
Simple design High enrichment Easy to multiplex targets Easy to multiplex samples High scalability Single reaction needed |
Erases native DNA modifications Limited fragment length Laborious protocols with high hands-on time Includes multiple PCR steps |
Cas-mediated enrichment | Up to 100 kb | 50–1,000× | 4.6%55 | 10 genes55 | ONT, PacBio |
Preserves DNA modifications Long read lengths No PCR |
High DNA input Less ability to multiplex |
Adaptive sampling | 10–20 kb | 15–40× | ~5%68 | 717 genes68 | ONT |
Simple design Preserves DNA modifications Long read lengths No PCR No additional molecular biology steps besides library prep |
Low enrichment Limited to ONT nanopore sequencing Multiple sequencing runs to obtain maximum output Computationally intensive |
ONT, Oxford Nanopore Technologies; PacBio, Pacific Biosciences.