Illuminating the Artefacts of NGS Seen in PGT-A & PGT-SR

Abstract

Background: Advancements in next generation sequencing (NGS) based genomics over the past 10 years have allowed the routine adoption of preimplantation genetic testing for aneuploidy (PGT-A), and/or segmental rearrangements (PGT-SR) of [Formula: see text]10Mb 1 in the assisted reproductive technology (ART) clinic. However, one challenge remains within the assessment of aligned short read sequencing data, in the ability to discriminate between embryonic profiles with genuine pathological copy number variations (CNVs) from artefacts arising from erroneous DNA amplification or library preparation. Aim: To identify the common artefacts seen in next generation sequencing (NGS) based PGT to reduce inconclusive or false positive results. Method: NGS profiles generated from biopsied human embryonic trophectoderm cells were manually analysed for common genomic artefacts. Following trophectoderm biopsy within a clinical ART cycle, cell samples underwent DNA amplification (Illumina SureplexTM DNA amplification system), sequencing (VeriSeqTM PGS kit on the MiSeq[Formula: see text] System, and bioinformatic data analysis (Bluefuse Multi v4.4, Illumina). Results: Common artefacts were identified affecting chromosomes 7, 11, and 19. Artefacts imitated small CNVs typically displaying a [Formula: see text]50% increase in centromeric sequence counts on chromosomes 7 and 11, and a global increase of sequence counts of [Formula: see text]40% across the entire chromosome 19 (except at the centromere, presenting as diploid). Interestingly a technical repeat of the library preparation and sequencing of the original DNA template somewhat normalised these artefacts. Conclusion: The study found three commonly occurring artefacts involving chromosomes 7, 11, and 19 artificially introduced during suboptimal NGS library preparation. Importantly these artefacts may be normalised through technical repeat of library preparation and sequencing. The awareness of these artefacts may reduce false positive and/or inconclusive results, increasing clinical embryonic utilisation following PGT.