Webinar: A High-throughput, High-resolution PGT-A Workflow with Polyploidy and Contamination Detection

Webinar summary

In this webinar, Adam Harris presents the development and evaluation of a high-throughput, high-resolution preimplantation genetic testing for aneuploidy (PGT-A) workflow implemented on the Ion Torrent Genexus Integrated Sequencer. The presentation focuses on analytical performance of the ReproSeq PGS workflow together with a newly developed polyploidy panel designed to provide SNP-based assessment of polyploidy, maternal contamination, and sibling quality control. 

Study overview

The study addresses analytical challenges associated with PGT-A, where genomic analysis is performed using small numbers of cells obtained from day 5 trophectoderm research samples. Whole genome amplification is required to generate sufficient DNA for comprehensive genomic analysis.

The ReproSeq PGS workflow combines SingleSeq whole genome amplification and library preparation with automated sequencing on the Genexus platform. The workflow supports high-throughput analysis while maintaining high-resolution copy number variant (CNV) detection.

In addition to conventional PGT-A analysis, the study introduces a targeted SNP-based polyploidy panel generated from split whole genome amplification products. This secondary workflow enables evaluation of:

• Polyploidy status
• Maternal contamination
• Sibling relationship quality control

Analytical validation was performed using characterized genomic DNA reference materials and cell line samples containing known CNVs, polyploid genomes, and controlled contamination models.

Key findings

High-throughput PGT-A workflow on the Ion Torrent Genexus System

• The Ion Torrent Genexus GX5 chip supports four independently operated sequencing lanes
• Each lane accommodates up to 48 libraries, enabling analysis of up to 192 libraries per chip
• The workflow combines automated sequencing with integrated whole genome amplification and sample barcoding

High-resolution CNV detection

• Analytical evaluation used purified genomic DNA diluted to approximately two-cell DNA input levels
• Verification focused on detection of approximately 10 Mb CNVs
• Across 288 evaluated libraries:
  • 284 expected CNVs were correctly identified
  • Four false negatives were observed
  • No false-positive CNVs were detected
• Overall analytical performance demonstrated:
  • 98.6% sensitivity
  • 100% specificity for targeted CNV detection

SNP-based polyploidy detection

• The polyploidy panel analyzes SNP allele frequencies following split whole genome amplification
• Diploid samples demonstrated the expected heterozygous allele frequency distribution centered at approximately 50%
• Triploid samples produced characteristic bimodal allele frequency distributions centered near 33% and 67%
• Triploidy calls additionally required detection of tri-allelic microhaplotype sites and elevated estimated contamination signals

Maternal contamination assessment

• Maternal contamination was evaluated through analysis of tri-allelic microhaplotype sites
• The workflow successfully detected experimentally introduced maternal DNA contamination at approximately 20–30%
• Estimated contamination levels provided quantitative guidance while distinguishing contaminated from uncontaminated samples

Sibling relationship quality control

• SNP-derived microhaplotypes enabled evaluation of expected familial relationships among embryo samples
• Hierarchical clustering and relatedness analysis correctly grouped sibling samples according to known family assignments
• The workflow provided an additional quality control measure to identify potential sample mix-ups

Integrated SNP analysis expands PGT-A information content

• Standard PGT-A cannot distinguish female triploid samples (69,XXX) from diploid female samples using copy number analysis alone
• Addition of SNP analysis enables identification of:
  • Diploid samples
  • Triploid samples
  • Haploid samples
  • Maternal contamination
  • Sample relatedness
• These complementary analyses extend the information generated from a single whole genome amplification workflow

This study demonstrates a high-throughput PGT-A workflow on the Ion Torrent Genexus System that combines automated sequencing with high-resolution CNV detection. Analytical evaluation showed strong performance for approximately 10 Mb CNV identification while supporting large-scale sample throughput.

Integration of a complementary SNP-based polyploidy panel further expands analytical capability by enabling assessment of polyploidy status, maternal contamination, and sibling quality control from the same amplified material. Together, these workflows provide an integrated genomic analysis strategy for preimplantation genetic testing research.