Improved short nascent strand sequencing (iSNS-seq) enhances DNA replication origin detection and reduces non-origin biases
Identifying DNA replication origins (ORIs) in human and other metazoan genomes has been challenging, as highlighted by the fact that various methods for mapping origins have produced conflicting results. A popular method, short nascent strand sequencing (SNS-seq), isolates newly replicated short single-stranded DNA by size selection and enriches them using $\lambda$ exonuclease ($\lambda$-exo). Surprisingly, SNS-seq has never been validated in  \textit{Saccharomyces cerevisiae}, where ORIs have been well characterized genome-wide. We improved the SNS-seq protocol and compared its origin-mapping sensitivity and specificity with traditional SNS-seq in asynchronous populations of \textit{Saccharomyces cerevisiae}. The improved SNS-seq protocol enhanced the enrichment of origin-derived SNS DNA. Strikingly, the traditional SNS-seq failed to detect known ORIs and instead enriched non-origin DNA, likely arising from RNA:DNA hybrids. These findings have important implications for the interpretation of previously published datasets that rely on $\lambda$-exo for ORI mapping. Overall, our biochemical and genomic analyses help unravel the mystery of the inconsistencies between SNS-seq and other techniques used to map DNA replication origins genome-wide.