Multiscale sequencing of G/C rich repeated genomic DNA via single molecule manipulation in magnetic tweezers – Museq

By design, most of the current DNA sequencing technologies rely on the use of an amplification step via a DNA polymerase. This fundamental step has two direct consequences. First, the layer of information carried by modifications on nucleotides in the original sample is lost. Although some workarounds are possible, they are indirect and costly. Second, certain regions, in particular guanine-rich repeated sequences, are difficult to sequence via polymerase-based methods. In addition, the short reads generated by current high throughput sequencing methods are ill-adapted for resolving repeated sequences, as they pose obvious computational problems during alignment to reference genomes. For these reasons, repeated sequences are often discarded from analysis, despite their potential importance for understanding genome composition and structure.
One of the few sequencing techniques which does not rely on de novo nucleotide incorporation is sequencing by oligonucleotide annealing using magnetic tweezers, which has been developed by the team of Vincent Croquette at the ENS lab. This method has several advantages to circumvent some limitations of current Next Generation Sequencing methods, and the aim of this project is to build on these features to develop an inexpensive, reliable method to sequence difficult G/C rich repeated sequences both at the genetic and epigenetic level.
The consortium of this project is composed of two academic partners (the ENS and the Museum) and one industrial partner (PicoSeq), which is a spin-off from the ENS lab and is developing the magnetic tweezers technology for the market. The consortium has already accumulated several results justifying the aims of this project. Over the last few years, Partner 1 (Museum) and Partner 2 (ENS) have collaborated on sequencing tandem-repeated DNA molecules with high G/C content, a type of sequence challenging for most sequencing techniques. As of today, they have successfully sequenced a 1kb molecule of a 52 base-pair G/C rich human minisatellite, which is beyond the reach of most current sequencing technologies. In addition, partners 2 and 3 (PicoSeq) have developed proof of principle experiments showing that the method of sequencing by oligonucleotide hybridization can be modified to identify epigenetic modifications on the DNA, by replacing oligonucleotides with antibodies to locate for a specific modification on the DNA molecule.
Now, the main challenge for this technology is to develop a reliable method to sequence specific loci, both at the sequence level and at the epigenetic level, of native DNA molecules captured from genomic samples, without relying on destructive PCR amplification steps. The three partners will work respectively on the following tasks:
-developing molecular biology methods to selectively capture a given G/C rich genomic locus from native genomic DNA samples, and process them in vitro to a molecular form amenable to manipulation by magnetic tweezers.
-developing of the sequencing technology to tackle to the analysis of G/C repeated sequences, by improving both oligonucleotide design (reducing their size, modifying chemistry and composition) and software tools. The goal is to extend the technology towards de novo sequencing, in particular handling secondary structures like G-quadruplexes.
-developing methods and reagents to sequence epigenetic modifications, with a focus on oxidation marks carried by guanines. Indeed, guanine oxidations is recognized as important biomarkers for aging and cancer, but their detection is still indirect and expensive by existing epigenetic sequencing methods.
Our project will provide the community with a tool for genetic and epigenetic sequencing of any genomic locus, including regions containing complex repeats. This is of interest for medical applications as well as fundamental aspects of genome dynamics.