NET-Seq
Pu-Seq
Pu-seq provides direct replication-origin location and efficiency data, as well as indirect estimates of replication timing. Alkali treatment of duplex ribonucleotide-containing DNA results in phosphate-backbone cleavage 3’ to the ribose, resulting in a 5’-hydroxyl end. If the denatured DNA is used as a template for random-hexamer primer extension, 5’ to 3’ synthesis results in a flush end adjacent to the initial ribose. By generating a library from single-stranded DNA and placing distinct index primers at each end, sequencing reads can be mapped to individual strands, to determine the original ribonucleotide position with single-base accuracy.
Pros:
- Single-base accuracy
Cons:
- Only applied to yeast