Comparative Analytical Evaluation of Two NGS-Based PGT-A Assays: ReproSeq vs VeriSeq

This presentation reports a systematic, empirical comparison of two widely used next-generation sequencing (NGS)–based assays for preimplantation genetic testing for aneuploidy (PGT-A)—VeriSeq PGS Kits (Illumina/Vitrolife) and Ion ReproSeq PGS Kits (Thermo Fisher Scientific). The study was conducted by Adam Goodman, Director of Preimplantation Genetics at NextGen MDx, a high-volume U.S. genetics laboratory with extensive experience across multiple PGT-A platforms.

The objective of the study was to determine whether differences in sequencing chemistry, library preparation, automation, and data analysis workflows between the two assays result in meaningful differences in analytical performance. Key evaluation criteria included resolution, accuracy, precision, reproducibility, data quality assessment, and operational scalability. The study design intentionally emphasized real-world sample conditions rather than idealized validation inputs.

Resolution assessment

Analytical resolution was evaluated using ten commercially available cell lines containing known chromosomal abnormalities ranging from whole-chromosome aneuploidies to small segmental copy number variants (as small as 1.5 Mb). Intact cells were diluted to biopsy-equivalent inputs (~5–10 cells) and processed multiple times through both platforms. Both assays consistently detected segmental events down to approximately 5 Mb, exceeding manufacturer-stated specifications. Copy number profiles generated by ReproSeq and VeriSeq were indistinguishable across all tested samples.

Accuracy and concordance

Accuracy was assessed through a blinded concordance study involving embryos previously classified as aneuploid using an SNP-array–based platform. Both original trophectoderm biopsies and corresponding whole embryos were reanalyzed using both NGS assays. ReproSeq and VeriSeq Kits demonstrated complete concordance with each other at the euploid versus aneuploid classification level, as well as full concordance between biopsy-level and whole-embryo results. A subset of embryos previously labeled aneuploid by SNP arrays (~20%) were identified as euploid by both NGS platforms, suggesting limitations in array-based methods rather than biological mosaicism.

Precision, reproducibility, and mosaicism

Assay precision and reproducibility were evaluated using repeated analysis of a known ~40% mosaic trisomy sample across 24 replicates and by two independent technicians. ReproSeq Kits demonstrated stable read distribution, low inter-sample variability, and mosaic fraction estimates within ±10% of the expected value. These findings are consistent with known quantitative limits of mosaicism detection and support caution against rigid stratification of mosaic categories.

Workflow and data analysis considerations

While both assays exhibited equivalent analytical performance, ReproSeq Kits demonstrated advantages in workflow design, including flexible run sizes, earlier sample barcoding, reduced consumable usage, hazardous reagents, and significant reductions in hands-on technician time through Ion Chef automation. Data analysis using Ion Reporter software provided enhanced quality assessment tools and visualization while maintaining the need for expert manual review.

Key findings

• Analytical resolution
  • Both assays reliably detected segmental copy number variants down to ~5 Mb
  • Performance exceeded manufacturer-stated resolution thresholds
  • Copy number profiles were identical across platforms
• Accuracy and concordance
  • 100% concordance between ReproSeq and VeriSeq kits for euploid vs aneuploid calls
  • 100% concordance between biopsy-level and whole-embryo results
  • ~20% of SNP-array–classified aneuploid embryos were euploid by both NGS assays
• Precision and reproducibility
  • Mosaic fraction estimates within ±10% of expected values
  • Coefficient of variation for read counts <10%
  • Results reproducible across technicians and sequencing runs
• Mosaicism interpretation
  • Observed variability supports reporting observed values rather than fixed mosaic categories
  • Findings consistent with known biological and technical detection limits
• Workflow and operational impact
  • Equivalent total assay duration (~12 hours)
  • ReproSeq kits enable automation, reduced hands-on time, and scalable run sizes (16–96 samples)
  • Reduced consumables, no hazardous reagents, and lower cumulative operational costs