HindIII treatment to enrich for mutations at restriction site. Specifically, after extracting DNA from the sperm of the donors, each sample is enriched for the APOL1 G1 1024A>G mutation and nearby mutations by the restriction enzyme HindIII, which cleaves the wild-type (WT) sequence AAGCTT at positions 1023-1028 of APOL1 while leaving the 1024A>G mutant and other mutants in these positions intact. Next, unique barcodes are attached to the DNA fragments from these HindIII-treated samples in order to reduce error by consensus sequencing of copies originating from the same original fragment.
Genomic DNA digested by HindIII (mutation enrichment enzyme) and PvuII (barcoding enzyme). Barcode is synthesized from PvuII digested site via fill-in reaction using APOL1 specific oligo. 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