PT  - JOURNAL ARTICLE
AU  - Magini, Pamela
AU  - Mingrino, Alessandra
AU  - Gega, Barbara
AU  - Mattei, Gianluca
AU  - Semeraro, Roberto
AU  - Bolognini, Davide
AU  - Mongelli, Patrizia
AU  - Desiderio, Laura
AU  - Pittalis, Maria Carla
AU  - Pippucci, Tommaso
AU  - Magi, Alberto
TI  - Third Generation Cytogenetic Analysis (TGCA): diagnostic application of long-read sequencing
AID  - 10.1101/2021.08.13.456226
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.08.13.456226
4099  - http://biorxiv.org/content/early/2021/08/15/2021.08.13.456226.short
4100  - http://biorxiv.org/content/early/2021/08/15/2021.08.13.456226.full
AB  - Unbalanced Structural Variants (uSVs) play important roles in the pathogenesis of several genetic syndromes. Traditional and molecular karyotyping are considered the first-tier diagnostic tests to detect macroscopic and cryptic deletions/duplications. However, their time-consuming and laborious experimental protocols protract diagnostic times from three to fifteen days. Long read sequencing approaches, such as Oxford Nanopore Technologies (ONT), have the ability to reduce time to results for the detection of uSVs with the same resolution of current state-of-the-art diagnostic tests.Here we compared ONT to molecular karyotyping for the detection of pathogenic uSVs of 7 patients with previously diagnosed causative CNVs of different sizes and allelic fractions. Larger chromosomal anomalies included trisomy 21 and mosaic tetrasomy 12p. Among smaller CNVs we tested two reciprocal genomic imbalances in 7q11.23 (1.367 Mb), a 170 kb deletion encompassing NRXN1 and mosaic 6q27 (1.231 Mb) and 2q23.1 (408 kb) deletions. DNA libraries were prepared following ONT standard protocols and sequenced on the GridION device for 48 h. Data generated during runs were analysed in online mode, using NanoGLADIATOR.We were capable to identify all pathogenic CNVs with detection time inversely proportional to size and allelic fraction. Aneuploidies were called after only 30 minutes of sequencing, while 30 hours were needed to call CNVs < 500 kb also in mosaic state (44%). These results demonstrate the clinical utility of our approach that allows the molecular diagnosis of genomic disorders within a 30 minutes to 30 hours time-frame.Competing Interest StatementThe authors have declared no competing interest.