Bsu36I treatment to enrich for mutations at restriction site. Specifically, after extracting DNA from the sperm of the donors, each sample is enriched for the HbS mutation and nearby mutations by the restriction enzyme Bsu36I, which cleaves the wild-type (WT) sequence CCTGAGG at positions 16–22 of HBB and the homologous positions of HBD while leaving the HbS mutant and other mutants in these positions intact. Next, unique barcodes are attached to the DNA fragments from these Bsu36I-treated samples in order to reduce error by consensus sequencing of copies originating from the same original fragment. : Sample EUR4_Bsu36I_treated
Genomic DNA digested by Bsu36I (mutation enrichment enzyme) and HpyCH4III (barcoding enzyme). Barcode is synthesized from HpyCH4III digested site via fill-in reaction using HBB/HBD specific oligos. Following multiple cycles of linear amplification of the barcoded strands using phosphorothioate containing primers, non amplified DNA molecules are degraded by 5' exonucleases. A second barcode is added to the other edge of the linearly amplified molecules via a single primer extension reaction and after removal of the excess unbound primers by ExoI exonuclease the barcoded ds DNA is amplified by PCR. Following NGS, de novo mutations are called at the sequence analysis stage by generating a concensus sequence for the target DNA strands