No Bsu36I treatment for the calculation of Bsu36I-rnrichment factor. Specifically, To determine how many target WT sequences have been removed (scanned) by Bsu36I digestion and hence to be able to calculate the de novo mutation rate, we run a second reaction in parallel for each donor. This reaction involves the same steps as the former reaction except for digestion by Bsu36I (Bsu36I-untreated). Spiking into the Bsu36I-treated and untreated samples known amounts of mock HBB-like DNA molecules that are fully resistant to Bsu36I digestion allows to calculate in the sequence analysis stage the Bsu36I-enrichment score based on the change in their frequency between the two treatments, which in turn is used to calculate the number of scanned WT sequences. : Sample AFR6_Bsu36I_untreated
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