Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

Next-generation sequencing has enabled the genome-wide mapping of human DNA replication origins. However, the results obtained using two independent approaches to map replication origins, namely sequencing isolated small nascent DNA strands (SNS-seq) and sequencing replication bubbles (bubble-seq), show only limited concordance. To address this controversy, we describe here an independent origin mapping technique that we call initiation site sequencing (ini-seq)..In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP in a cell-free initiation system near the sites of its initiation. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify more than 25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq in this way are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. We find that sites identified by ini-seq show greater concordance with sites identified by SNS-seq than with those identified by bubble-seq, and they overlap with positive nucleotide distribution skew jumps in the genome more frequently than do sites identified by either alternative technique. Overall, our novel origin mapping technique complements and consolidates alternative methods and shows greater specificity than either.