SV detection and analysis workflow with an example of complex rearrangement. (A) We used Manta to call SVs in our fetal brain clones. Each clone was compared to all other clones, and germline events were filtered out based on multiple recurrence in the clone-to-clone comparison (Methods). For the resulting high-confidence SVs, we implemented assembly and genotyping workflows as a further in silico validation. (B) Example of mosaic complex SV detected. This intra-chromosomal rearrangement on Chromosome 12 (intronic in NAV3 gene) was detected in subject 316 clone #19 from basal ganglia (BG) and involves two deletions, one inversion, and one duplication. Based on the contig sequence generated from our assembly analysis, we hypothesize a replication model as depicted by arrows in the top panel (dashed lines and arcs represent hypothesized replication fork switches). Pairwise alignment of the contig against the reference sequence confirmed the SV and defined the breakpoints of rearrangements (colored arrows in the box represent aligned segments). Using two sets of forward and reverse primers indicated by block blue (Ref) and brown arrows (Alt), polymerase chain reaction (PCR) validated the SV in this clone (results for the second set of primers are represented in Supplemental Fig. S3). Blue arrow: duplication; yellow arrow: inversion; cyan and purple segments: deletions.
