New protocol based on massive parallel sequencing for aneuploidy screening of preimplantation human embryos

Abstract

Novel next-generation sequencing procedures have rapidly emerged into the preimplantation genetic screening framework. This work presents the design and validation of a new low-coverage whole-genome sequencing assay for aneuploidy detection in single blastomeres and trophectodermal samples from preimplantation embryos. The validation ensures analytical sensitivity, specificity, robustness, precision, limit of detection, resolution, and reproducibility. Specific parameters to measure the performance are defined, and the results are compared with a standardized array-based method to stablish the concordance. From the single cell genomics point of view, the main novelties are the length of reads of the libraries (150 nucleotides) together with a paired-end strategy and the design of an original algorithm and copy number viewer. A total of 129 samples were included in six experimental runs using a MiSeq Illumina platform. Samples included: single amniocytes, single blastomeres (cleavage-stage embryos), trophectoderm samples (blastocyst), and diluted DNA. Sensitivity and specificity were calculated per chromosome yielding 96% and 99%, respectively. The percentage of concordant samples was 98.2% and all of the aneuploid samples were confirmed. In conclusion, the validation yields highly reliable and reproducible results, representing an accurate and cost-effective strategy for the routine detection of aneuploidy in human embryos.

Segmental aneuploidy in human blastocysts: a qualitative and quantitative overview

“…Several platforms and protocols based on NGS have been validated [8–15], and can be employed to assess altered distribution patterns of segmental aneuploidies. Previous studies using NGS technology have described a minimum size of almost 14 Mb for detecting imbalances [9, 16, 17], although other authors have reported imbalances of 1.5–1.8 Mb [14, 15] and segmental aneuploidies as small as 10.0 Mb [18].…”

Second-generation preimplantation genetic testing for aneuploidy in assisted reproduction: a SWOT analysis

“…The appearance of new technologies, such as NGS, could help to significantly reduce the costs of PGT-A while retaining a high level of accuracy of detection compared with the existing techniques. These are essential for PGT-A 2.0 to be widely adopted (Chen et al 2015: evidence level 1a;Huang et al 2016: evidence level 4) and represent a cost-effective strategy for the routine detection of aneuploidy in human embryos (Vendrell et al, 2017).…”