MLOF F8 and F9 MIP next-generation DNA sequencing strategy. Schematic of the MIP targeted sequencing of the F8 and F9 genes in hemophilia. First, MIPs were designed to capture all coding, 5′, and 3′ untranslated regions (UTRs) of both the F8 and F9 genes. Additional MIPs were designed to detect unique sequences produced when genomic DNA carrying one of the common F8 intron 1 or intron 22 inversions is digested with Ksp22I and ligated as well as the normal reference, or wild-type, F8 sequence. Genomic and Ksp22I digested and ligated DNA was prepped and mixed with pooled dephosphorylated MIPs, 2′-deoxynucleoside 5'-triphosphates (dNTPs), polymerase, and ligase for MIP target gap filling and ligation. MIPs were released by exonuclease digestion, and the library was individually bar-coded, pooled, and sequenced using an Illumina MiSeq or NextSeq (schematic adapted from O’Roak et al¹⁶ ). The resulting DNA sequence data were cleaned, subjected to quality control filters, aligned to the reference genome (hg37), and annotated for analysis in the clinical laboratory.