Figure 2

Identification and characterization of a BEST1 deletion using targeted locus amplification (TLA) on extracted genomic DNA. (a) Location of the deletion. The heterozygous 8-kb deletion identified in P1 is depicted in red, removing the first two exons of the BEST1 gene. (b) Primer design TLA. Initial real-time polymerase chain reaction (qPCR) delineation of the deletion was hampered by highly repetitive breakpoint regions, followed by further delineation using TLA. The minimal deleted region based on the qPCR delineation is depicted in red; the gray dotted line corresponds to the 5′ and 3′ breakpoint regions and the surrounding genomic region in black. In case of a deletion, the outward-oriented TLA primers indicated in orange need to be designed in an anchor region that is not deleted, here in the downstream surrounding genomic region. (c) TLA read mapping. The amplified PCR products are sequenced by next-generation sequencing (NGS), followed by mapping of the resulting reads showing one distinct peak surrounding the anchor region. (d) Delineation of the deletion. Detailed analysis of the reads in the breakpoint regions led to the identification of the deletion at nucleotide level: chr11:g.61711373_61719810del. The upper, middle, and lower lines represent the sequences of the 5′ wild-type (wt) region, the deletion junction product, and the 3′ wt region, respectively. Microhomology of 3 bp is indicated in gray. The two boxes show the mapped reads in both breakpoint regions. Green sequences are forward reads; red sequences are reverse reads. The fully colored reads are wt reads, while the reads containing shaded sequences are junction reads spanning the deletion breakpoints. The turquoise and orange boxes highlight the 5′ and 3′ sequences, separated by the 3 bp of microhomology.