title: Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq).
abstract: Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >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 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. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq.
authors: Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, London NW7 1AA, UK.
authors: Alexander R
authors: AR
authors: Langley
authors: Department of Medicine, University of Cambridge, Cambridge CB2 0QQ, UK.|Department of Haematology, University of Cambridge, Cambridge CB2 0PT, UK.
authors: Stefan
authors: S
authors: Gräf
authors: Francis Crick Institute, Mill Hill Laboratory, The Ridgeway, London NW7 1AA, UK.
authors: James C
authors: JC
authors: Smith
authors: Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK tk218@cam.ac.uk.
authors: Torsten
authors: T
authors: Krude
mesh_terms: D045744:Cell Line, Tumor; D002874:Chromosome Mapping / Q000379:methods; D019295:Computational Biology / Q000379:methods; D004247:DNA; D004261:DNA Replication; D015894:Genome, Human; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D059372:Nucleotide Motifs; D018741:Replication Origin; D017422:Sequence Analysis, DNA; D024363:Transcription Initiation Site
chemical_list: C022262:Okazaki fragments; D004247:DNA