Spotlight on the artifacts in next-generation sequencing in PGT-A: Reason for high mosaicism reporting

Abstract

The study investigates artifacts in Next-Generation Sequencing (NGS) used for Preimplantation Genetic Testing for Aneuploidy (PGT-A) and their contribution to the high rates of mosaicism reporting. Modern PGT-A can detect mosaicism by analyzing copy number variations (CNVs) in embryonic biopsies, yet distinguishing true mosaicism from artifacts remains challenging. In a cohort of 22 embryos, NGS profiles revealed recurring artifacts on chromosomes 7, 11, 16, and 19. These artifacts likely result from errors in DNA amplification for NGS library preparation, potentially leading to false mosaicism diagnosis. The study utilized DNA extracted from trophectoderm biopsies, spent culture media, and whole blastocyst samples, with CNV analysis performed using BlueFuse Multi software. Quality control parameters such as DLR noise, read count, and quality score were considered, confirming that technical inconsistencies contribute to the observed artifacts. Findings align with prior research, suggesting the need for improved NGS protocols to minimize these errors. Enhanced internal validation and adoption of new technologies could reduce false-positive rates and improve clinical decision-making in PGT-A.