Toward Small CNV Detection in NIPT: A Preliminary Analytical Evaluation of Sequencing Depth and Workflow...

Background/Objectives: Non-invasive prenatal testing (NIPT) performance is influenced by multiple biological and technical factors, including fetal fraction, sequencing depth, library preparation, and bioinformatic processing. This study aimed to compare two NIPT workflow configurations based on Vazyme and Illumina platforms and to evaluate the potential impact of sequencing depth and workflow design on analytical performance. Methods: A total of 1000 maternal plasma samples were analyzed using two multiplexing configurations (24-plex for Vazyme and 48-plex for Illumina). A workflow-specific reference baseline was constructed using 700 clinically low-risk samples presumed to be euploid to optimize normalization procedures and CNV detection. In addition, a synthetic 22q11.2 deletion control was evaluated across different fetal fraction levels to assess analytical feasibility for small CNV detection. Results: The 24-plex workflow generated a higher number of reads per sample (~18M vs. ~9M) and showed reduced analytical variability, particularly in low fetal-fraction samples. Failure rates were lower in the 24-plex workflow (<1% vs. >2.5%). Baseline optimization improved normalization and signal stability. The synthetic 22q11.2 deletion control (~2.5-3 Mb) was consistently detected at fetal fractions ≥6%, supporting the analytical feasibility of small CNV detection under controlled experimental conditions. Conclusions: Higher sequencing depth combined with robust baseline construction may improve NIPT analytical performance and enhance sensitivity for subchromosomal CNV detection. However, the present study should be regarded as a preliminary analytical feasibility study rather than a clinical validation study. Additional prospective investigations including clinically confirmed CNV cases are required to establish the true clinical performance of this approach.