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. 2023 May 9:1–15. Online ahead of print. doi: 10.1038/s41576-023-00600-1

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.