Figure 4
NIPT for sickle cell disease and cystic fibrosis by amplicon NGS. (A) Target region of HBB exon 1 NIPT and alignment with HBD. Start codon is shown as ‘M’. rs713040 is a benign variant with high allele frequency. rs33930165 and rs334 are pathogenic variants. (B) Target region for CFTR NIPT of the F508del variant. (C) The NIPT assay uses pathogenic allele fraction, fetal fraction, and assayed genomic equivalents data to compute a likelihood ratio (LR) for an affected homozygous vs heterozygous fetus. Regions where \(\frac{1}{8} < {\rm{LR}} < 8\) are shaded white to represent no-call regions. Affected NIPT calls are shaded blue, and non-affected calls are shaded red. (D) Validation of allele fraction measurement for sickle cell disease. Genomic DNA corresponding to a heterozygous sickle cell trait (NA20838) mother was sheared to 150 bp and 0 to 10% sheared sickle cell disease DNA (NA16265) was mixed in. The HbS allele fraction (AF) was measured by amplicon NGS sequencing. When no SCD DNA is mixed in, the mean HbS AF = 0.501 (n = 80, CV = 1.8%). With 10% SCD DNA, mean HbS AF = 0.563 (n = 80, cv = 1.6%). QCT analysis measured 2200 GE in these assays. The total number of samples shown is n = 184. (E) Validation of allele fraction measurement for cystic fibrosis. Sheared homozygous cystic fibrosis variant F508del was spiked into 30 ng heterozygous F508del sheared DNA at the indicated proportions. F508del MAF was calculated with the following formula \(\frac{{D}_{F508}}{{D}_{wt}+{D}_{F508}}s\), where s is a shearing correction factor. The shearing correction factor compensates for the increased presence of the 3 bp shorter F508del allele present in sonicated DNA. The correction factor is derived from a geometric distribution of sheared fragments with a mean length of 150 using the following formula \(s={(1-\frac{1}{150})}^{100}/{(1-\frac{1}{150})}^{97}=0.980\).
