Massive genotyping of structural variants by analysis of juxtaposed sequence pairs in cis in the restriction circles. Physical selection of genomic regions and development of new ad hoc algorithms.

Numerous approaches to genomic analysis were developed at the Lab-GMH. Among them, in 2004 the method of inverse shifting-PCR (IS-PCR) was designed, developed and validated for Inv22 genotyping. Currently, IS-PCR is the method of choice in most hemophilia laboratories in the world to investigate the Inv22. This IS-PCR approach was experimentally optimized and extended in its application to encompass Inv1 genotyping and all the complexity of int22h-associated rearrangements (i.e., Inv22 type 1 and 2, and variants, Del22 and Dup22). With the same principle of specific amplification from the ends of a circularized restriction fragment, extending the limits of the product to include highly informative genetic markers (eg, STRs, SNPs, RFLPs), we have developed the PI technique (phasing by inverse-PCR) that allows determining the haplotype phase in cis of these markers 1-30 kb apart. Among other potential applications in human genetics and evolutionary genetics, this methodology has opened new perspectives for the prenatal and preimplantational diagnosis of prevalent F8 inversions through the use of surrogate polymorphic markers obtained directly by the PI technique, avoiding the need to carry out linkage analysis in the family.

Currently, the principle of restriction circle analysis to match distant sequences of the same molecule is being implemented coupled to massively parallel sequencing (MPS) with the key objective of providing additional long-distance information for massive genotyping of structural variants (SV) (eg, large deletions, duplications, and inversions), either mediated by NAHR between duplicons or not. The experimental challenges that arise are the optimization of the procedures of fragment circularization and specific circles capture, by means of padlock probes, and the development of the bioinformatic algorithms necessary to carry out the genotyping of SVs integrating the information of the paired end sequences of the MPS reads that contain a restriction site. Pilot studies are focused on studying the F8 inversions, in particular the elusive Inv22.