Improved short nascent strand sequencing (iSNS-seq) enhances DNA replication origin detection and reduces non-origin biases

Identifying DNA replication origins in human and other metazoan genomes has been challenging, as highlighted by the fact that various methods for mapping them have produced conflicting results. A popular method, short nascent strand sequencing (SNS-seq), enriches newly replicated short single-stranded DNA by size selection and λ -exonuclease ( λ -exo) digestion of parental DNA. Surprisingly, SNS-seq has never been validated in Saccharomyces cerevisiae where origins have been well characterized genome-wide. Here we improved the SNS-seq protocol through biochemical optimization and benchmarked its origin-mapping sensitivity and precision against traditional SNS-seq in asynchronous populations of S. cerevisiae , a genetically tractable system that allows direct comparison against a well-defined set of confirmed origins. Relative to traditional SNS-seq, the improved protocol substantially increased enrichment of origin-derived DNA. Strikingly, traditional SNS-seq failed to detect known origins 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 λ -exo for origin mapping, and provide a proof-of-concept benchmark for extending this improved protocol to metazoan systems. Furthermore, 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.