NanoPoRep

The project proposes a revolutionary method to characterize DNA replication on single molecules. Eukaryotes replicate DNA by activating numerous replication origins that each establishes two divergent replication forks that perform bi-directional DNA synthesis with variable speeds. Despite the advent of DNA micro-array and massive DNA sequencing technologies, origins remain difficult to identify and our knowledge is still incomplete. Solving the nature of origins and analyzing replication progression require high-throughput analyses at the single molecule level because cell-population methods provide only an average picture where inter-cellular variability and rare events are masked in the average. State-of-the-art single molecule methods monitor the incorporation of non-standard nucleotides during replication. However, they provide no sequence information unless they are combined with DNA probes. This is laborious, low resolution and low throughput. High-throughput single molecule genomic profiling of DNA replication remains to be achieved. This challenge can be attained using nanopore sequencing as the monitoring apparatus of non-standard nucleotide incorporation. Sequencing precisely localizes the analyzed molecules on the reference genome and promises high-throughput. Nanopore sequencing is an emerging technology where ionic current through a bioengineered nanopore is recorded during the transit of a single-stranded DNA molecule through the pore. The nucleotide sequence of the molecule is then determined from the resulting current signal variations thanks to dedicated signal processing tools. We carried out a pilot project providing a proof of concept that available nanopore technology sensitivity is sufficient to carry out the project. The main scientific barrier to be lifted is thus to make a nanopore current analysis pipeline discriminating at least one of the non-standard nucleotides compatible with DNA replication studies, in addition to the four canonical nucleotides. We will develop an open-source signal processing pipeline for nanopore current analysis concomitantly with experimental validations and applications. We will follow neural network based approaches that are currently the most efficient methods in the field. The first objective is to detect replication tracks resulting from one short pulse (1-2 minutes) of bromo-desoxyuridine (BrdU), a thymidine analog, allowing to measure replication initiation and progression much more precisely and at higher throughput than the state-of-the-art but also to immediately identify the genomic loci where replication events take place. The second objective is to make the technology also sensitive to replication progression orientation. We will tackle this challenge using two consecutive pulses of BrdU of different concentration and developing the analysis pipeline that can discriminate them. The spatial order of low and high BrdU incorporating regions will provide the replication progression orientation at each pair of BrdU tracks. NanoPoRep project will determine the distributions of single replication fork speed and orientation at all points in the genome of yeast and human cells with a high resolution, and characterize DNA replication stochasticity. NanoPoRep will question the relationship between replication progression and chromatin organization and provide maps of potentially asymmetric replication barriers. The final product delivered by NanoPoRep will both contain software usable by other teams to directly analyze nanopore replication datasets and a programming suite allowing readily adaptation to other experimental settings (different non standard nucleotide or nanopore). NanoPoRep will thus contribute to the emergence of nanopore sequencing as a technological breakthrough for biological assays and diagnostic tools and thus help to respond to challenges in biology and human health.