Limited sensitivity of NGS-based detection of unbalanced products from reciprocal translocations inpolar bodies and blastocysts

Abstract

Introduction Numerous publications have demonstrated that preimplantation genetic diagnosis (PGD) increases the implantation rate and percentage of healthy newborns or at least decreases themiscarriage rate for translocation carriers with recurrent pregnancy losses (Fisher et al. 2010, Ikumaet al. 2018). The aim of this study was to evaluate whether VeriSeq PGS, a next generation sequencingbasedmethod for aneuploidy screening, is also suitable for the detection of structural aberrations inpolar bodies and trophectoderm cells. Since the product provider didn´t validate the method forunbalanced structural changes, our goal was to determine its sensitivity. Material & methods Array-based preimplantation genetic diagnostic was performed on polar bodies obtained from 12 women as well as on trophectoderm samples from five couples being translocation carriers. Furthermore, polar bodies from four women carrying other structural aberrations (i.e.versions, insertions, duplications) were included in the study. Whole genome amplification (WGA)was performed using the SurePlex DNA Amplification System. Cy3-labelled sample DNA and Cy5-labelled male reference DNA were hybridized for 4-16 hours on 24sure+ arrays. The slides were washedand scanned according to the manufacturer´s protocol. NGS libraries (VeriSeq PGS, Illumina Inc.) wereprepared from the same WGA products and sequenced on a MiSeq as described by the manufacturer. Data obtained with both methods were analyzed using the BlueFuse Multi Software. Fisher's exact testwas applied for statistical analyses. Results Reciprocal translocations present on a total of 18 chromosomes were included in this study. The results obtained with next generation sequencing in general delivered an excellent signal-to- noiseratio and a high dynamic range. Although, no statistically significant differences in the detection ofstructural variants between aCGH and NGS were observed, the comparison of both methods revealedseveral discordant results. In three cases, only one out of two translocated segments was detectableby NGS, whereas the aCGH method identified both fragments. By contrast, in the cohort with otherstructural aberrations, a 17 Mb insertion of chromosome 3 was detected only by NGS. Conclusions Our data indicate that although it is possible to analyze structural aberrations in singlecells with VeriSeq PGS, this method has some limitations in the detection of small telomericimbalances. The detection of structural aberrations is not only determined by their size but moreimportantly, it is locus-dependent. Hence, unbalanced reciprocal translocations with both translocatedfragments smaller than 8 Mb containing repetitive or paralogous sequences, might not be detectablewith VeriSeq PGS resulting in false negative results. Therefore, PGD for such translocations requires aprevalidation by e.g. sequencing of single sperm cells or unbalanced products of conception in orderto determine the locus-specific detection sensitivity.