Table 1 Comparison of multiplexed, targeted DNA sequencing methods in plants
Method | Custom design flexibility | Read length | Example of sequencing depth | Example of on-target read % in plant genome | Example of target number | Cost | Adaptation in DNA methylation detection |
|---|---|---|---|---|---|---|---|
Multiplexed PCR | limited by low complexity of sequences and potentially methylated sites | Short (e.g., < 200 bp) [42] | 1,000x [42] | 50.9% [42] | 443 markers [42] | low | no |
Probe hybridization-based | limited by commercial panels and costly design | short/long (e.g., 3.5 kb) [43] | 55x [43] | 52.9% [44] | 190 genes [44] | high due to modified probes | no |
CRISPR-based | limited by PAM sites and cleavage efficiency | short/long (e.g. 8 kb) [45] | ∼ 200x [45] | 3.04% [45] | 1 gene [45] | high due to RNA oligos and specific proteins | no |
FENGC | cleavage for any specified sites | short/long (e.g., > 500 bp) | 10 − 5,000x | ∼ 90% | 102 genic regions | low, all reagents are available in market | long-read EM-seq |