Pu-seq
Polymerase Usage Sequencing
Pu-seq provides direct replication-origin location and efficiency data, as well as indirect estimates of replication timing (Daigaku et al., 2015).
Alkali treatment of duplex ribonucleotide-containing DNA results in phosphate-backbone cleavage 3ê to the ribose, resulting in a 5ê-hydroxyl end. If the denatured DNA is used as a template for random-hexamer primer extension, 5ê to 3ê 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.
Advantages:
- Single-base accuracy
Disadvantages:
- Only applied to yeast
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
None available yet
References:
Keszthelyi A., Daigaku Y., Ptasinska K., Miyabe I. and Carr A. M. Mapping ribonucleotides in genomic DNA and exploring replication dynamics by polymerase usage sequencing (Pu-seq). Nat Protoc. 2015;10:1786-1801