#249 : Spotlight on the Artefacts in Next Generation Sequencing in PGT-A: Possible Reason for High Mosaicism Reporting

Background and Aims: Advancement in Next Generation Sequencing (NGS) has led to the extensive utilization of Preimplantation Genetic testing for Aneuploidy. But one challenge, in reporting of aligned short read sequencing data, is the ability to discriminate between embryonic NGS profiles with genuine pathological copy number variations (CNVs) from artefacts arising from erroneous DNA amplification/library preparation. Do technical errors in VeriSeq-NGS lead to artefacts in CNV charts resulting in falsely increased reporting of chromosomal mosaicism? Method: After ethical clearance and informed consent, patients [Formula: see text] 35 years, [Formula: see text]1 implantation failures and severe male factor infertility underwent PGT-A. Those with aneuploid embryos on TE biopsy, donated their embryos for research in year 2022. The DNA extraction and amplification was carried out using Sureplex-DNA amplification system. The libraries were prepared using VeriSeqTM-PGS Library Preparation kit. A total of 24 libraries were pooled, denatured and subjected to NGS using Illumina MiSeq system. CNV visualization and analysis for each sample was carried out using BlueFuse Multi Software (Illumina, USA). The aneuploid whole blastocyst were subjected to the same protocol. Results: NGS profiles generated from the aneuploid whole blastocysts, were manually analyzed for CNVs and for any common genomic artifacts. The quality control was performed using Illumina-Quality Control requirements and only samples satisfying these requirements were used for analysis in the study. Common artifacts were identified affecting chromosome 7,11,16 and 19. Out of 22 blastocysts, NGS profiles of 21 blastocysts had artifact at chromosome 19, and 15 had artifacts at chromosome 7,11,16. These artifacts may be introduced during whole genome amplification and/or suboptimal NGS library preparation or is inherent weakness of the VeriSeq-NGS kit. Conclusion: The four commonly occurring artefacts involved chromosome 7,11,16 and 19, which could be introduced during steps of whole genome amplification or library preparation. The awareness of these artefacts is important to reduce the false positive/inconclusive rate.