Etude Prospective Comparative Des Performances de Détection Des Variations de Nombre et de Structure Des Chromosomes Par Les Techniques de Cartographie Moléculaire et de Séquençage de Grands Fragments

Chromosome aberrations are found in up to 1% of the general population. Structural aberrations, either balanced (3.6‰) or unbalanced (0.9‰), represent a third of them. Most (but not all) unbalanced anomalies are associated with a relevant phenotype, while most (but not all) balanced rearrangements have no consequence on the phenotype except possible reproductive disorders. Indeed, the prevalence of the latter among infertile individuals is ten times higher than in the fertile population. Moreover, 6% - 27% of apparently balanced rearrangements can lead to developmental disorders through various mechanisms.

First tier chromosome analysis methods, karyotype and chromosomal microarray analysis (CMA), are hampered either by a low resolution (karyotype) or by their inability to detect balanced rearrangements (CMA). However, karyotyping is still the gold standard analysis in case of reproductive disorders (RD) and recurrent miscarriages because of the high prevalence of aneuploidies (mainly sex chromosome aneuploidies, i.e. 45,X or 47,XXY) or balanced structural abnormalities.

According to a French national annual survey carried out by the Agence de la Biomedecine, the diagnostic yield is ~5%-10% for karyotyping in RD and ~15-20% for CMA in ID/MCA. Hence, many patients remain without a molecular diagnosis of their condition after these first tier studies. Whole exome sequencing and now whole genome sequencing have been shown to be able to rise the diagnostic yield to up to 50-60% in ID patients. However, current short read sequencing methods fall short to providing robust data for structural variation (SV) detection, because of the inability of bioinformatic tools to map correctly the sequences due to the high proportion of homologous sequences at SV breakpoints. New emerging methodologies based on long DNA fragments are now available and may provide a way to circumvent current limitations: long read sequencing (lrNGS) and optical genome mapping (OGM) :

OGM has been developed by Bionano Genomics and combines microfluidic and high-resolution microscopy to offer an imaging of long, high molecular weight, DNA molecules (up to more than 1Mb) labelled with specific sequence tags. From these images, a de novo assembly of any patient's genome can be performed and compared to a reference genome map to unravel all kind of structural rearrangements with a resolution that is 100 to 1000 times higher than with karyotyping. A second pipeline based on coverage in each region allows for the detection of large CNVs and aneuploidies.

lrNGS of long DNA fragments (several kb) reduces short read sequencing based assembly issues due to repetitive sequences, and allow for detection of all mutations, from SNVs to SVs and CNVs. Due to the large size of fragment inserts, less false positive are expected for SV calling than observed with short read or linked-read sequencing. Although bioinformatics pipelines are improving, prospective detection of large SV/SNV remains challenging, with a very high rate of false positives and false negatives. Furthermore, no study has been conducted to test prospectively the feasibility and medical efficiency of using long read sequencing as a first-tier all-in-one test for SV and CNV identification.