Overview of next-generation sequencing. (A) Three separate primer pairs were designed to generate different amplicons that cover the HbS mutation site. (B) Fetal fractions were determined by the RASSF1A promoter methylation status, as described previously.⁹  The amount of fetal DNA was assessed by comparing the levels of amplification seen following methylation-sensitive restriction digests with amplification in corresponding undigested samples. For library preparation, single-plex PCRs were performed using 3 different primer pairs to mitigate the effects of any PCR biases that may occur with any individual primer pairs. Small primers were used in an initial limited 17-cycle PCR to preserve the wild-type to sickle allele ratio of the template. A second amplification was performed using longer primers that contained the relevant adapters, primer binding sites, and barcodes necessary for sequencing. As amplicon 3 consistently gave slightly higher yields than amplicons 1 and 2, only 22 cycles were required to generate enough library for sequencing, whereas 25 cycles were required for amplicons 1 and 2. Chr, chromosome.