LAUSR

Introduction Next Generation Sequencing (NGS) has entered in reproductive genetics for PGT-A with a great success. However, no solution for other alterations such as monogenic disorders and detection of balanced translocations has been developed. We have developed a complete workflow that can be run from a single biopsy. The workflow combines aneuploidy screening (PGT-A) with the additional analysis of monogenic disorders (PGT-M). For PGT- M, the test includes the direct detection of the mutation (when feasible) and the indirect test by analyzing several polymorphisms, previously selected by bioinformatic analysis, around the mutation. This test can be also applied to patients carrying balanced translocations. Material and methods Amplification and library preparation for PGT-A has been done using Ion Reproseq™ (Thermo Fisher). If its combination with PGT-M is required, an Ampliseq panel (Thermo Fisher) is developed for this gene. This includes the mutation of interest (when possible) and hundreds of selected (by in silico analysis) polymorphisms. These polymorphisms are selected in a way that can be used for any couple requiring this procedure. Both libraries (PGT-A and PGT-M) are combined and sequenced in a single run. The protocol allows the automation of many steps with the Ion Chef™ (Thermo Fisher), reducing hands-on time. For balanced translocations, a similar procedure is followed. Results Up to now, this solution has been developed for more than 60 monogenic disorders and applied tomore than 100 couples. The protocol has been validated with STR analysis and Sanger sequencing. Regarding to balanced translocations, one case study is in progress with promising results. The protocol developed works either manually (with results in 12 hours) and in a semiautomated way. Conclusions We have developed a complete protocol that allows the simultaneous analysis of PGT-A and PGT-M in a single NGS run.