Effect quantification and value prediction of factors in noninvasive detection for specific fetal copy number variants by semiconductor sequencing

Background The detection limit of noninvasive prenatal testing (NIPT) by next generation sequencing for any given fetal copy number variants (CNV) can be influenced by several factors. In this study, we quantified the effects and predicted the value of parameters for CNVs detection by NIPT. Methods Genomic DNA from patient's leucocytes with 3.16 Mb microdeletion in 22q11.21 was mixed with DNA from aborted fetal tissues without CNV at various concentrations by an enzyme digestion method. Abnormal DNA at 0% served as negative control. Sequencing of mixture samples (at 0%, 4%, 12%, and 20%) by Ion Proton Sequencer was performed at flow 500, with WISECONDOR as the pipeline in CNV-calling and bin of 500, 750 and 1,000 kb for counting unique reads. The parameters were evaluated with Box-Behnken design. The region with Z score <=-3 was marked as a potential microdeletion. Results The equation of Z score depending on fetal fraction, unique read number and bin size was obtained by Box-Behnken design. The negative effect was quantified as the coefficient in the equation. The smallest values of these parameters were defined as 4 M unique read number, and 10.08% fetal DNA concentration at bin of 750 kb for detecting subchromosomal microdeletion of 3.16 Mb. Conclusion The quantification of effect and value of parameters as well as the method used in this study can benefit the establishment of quality standards for CNVs detection and interpretation of CNVs detection results.