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

Abstract

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 {lambda}-exonuclease ({lambda}-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 {lambda}-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.