Preclinical validation of a targeted next generation sequencing-based comprehensive chromosome screening methodology in human blastocysts

Abstract STUDY QUESTION Can a novel targeted next generation sequencing (tNGS) platform accurately detect whole chromosome aneuploidy in a trophectoderm biopsy and provide additional information to improve testing? SUMMARY ANSWER Karyotypes obtained by tNGS were concordant with other validated platforms and single nucleotide polymorphism genotyping information obtained can be used for improved detection and quality control. WHAT IS KNOWN ALREADY qPCR-based whole chromosome aneuploidy screening is highly accurate in comparison to other common methods and has been shown to improve IVF success in two randomized clinical trials. With aneuploidy screening becoming standard of care in many IVF centres, there is a need to develop platforms with high throughput, low cost capabilities. STUDY DESIGN SIZE, DURATION Twelve well-characterized cell lines were obtained from a commercial cell line repository and 31 discarded human blastocysts were obtained from 17 IVF patients who underwent comprehensive chromosome screening (CCS). PARTICIPANTS/MATERIAL, SETTING, METHODS All samples were processed using a unique amplification strategy which directly incorporated sequencing library adapters and barcodes. Sequencing was performed on an Ion Torrent Proton. A custom bioinformatics pipeline was used to determine the karyotype for each sample. The consistency of tNGS diagnoses with either conventional karyotyping of cell lines or quantitative real-time PCR based CCS of blastocyst biopsies was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE Overall consistency per sample of tNGS based CCS in 5-cell samples from a variety of cell lines was 99.2%. In the blinded analysis of rebiopsies of aneuploid blastocysts, an overall targeted tNGS CCS consistency of 98.7% was observed per sample. These data demonstrate the ability of tNGS based CCS to provide an accurate and high throughput evaluation of aneuploidy in the human blastocyst. LARGE SCALE DATA Not applicable. LIMITATIONS REASONS FOR CAUTION This study is limited to whole chromosome aneuploidy, as mosaicism and segmental aneuploidy have not been investigated. WIDER IMPLICATIONS OF THE FINDINGS These data show an accurate, high throughput method, and with the greater depth of each amplicon sequenced in comparison to commercial kits, there is greater application available for single nucleotide polymorphism based analysis for quality control. STUDY FUNDING/COMPETING INTERESTS This study was funded through intramural research funds prov