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Keywords:

  • DNA < fetal cells;
  • nucleic acids and proteins;
  • trisomy 21;
  • trisomy 18;
  • aneuploidy;
  • next-generation sequencing;
  • cell-free DNA

ABSTRACT

Objective

To develop a novel prenatal assay based on selective analysis of cell-free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18).

Methods

Two hundred ninety-eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from average-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths.

Results

At the lowest sequencing depth, corresponding to 204 000 sequencing counts per sample, average-risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410 000 counts per sample substantially improved separation of aneuploid and average-risk cases. A further increase to 620 000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches.

Conclusion

Digital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment. © 2012 John Wiley & Sons, Ltd.