DNA Replication Errors Drive Genome‐Wide Small Inverted Triplication Dynamics

ABSTRACT Structural variants (SVs) have a profound impact on phenotype and diversity and are associated with human diseases. To explore the origination of SVs, we have analyzed 1,340 cancer genomes with annotation of 4,608 novel small inverted triplication (SIT) events and found that FEN1 is strongly associated with SIT incidence. Then, we performed long‐read sequencing and developed PacBioR to annotate SITs in yeast FEN1 mutant cells. We found that SIT structures mimic classic inverted triplications but with a smaller DUP/IN/DUP structure of 148/160/148 bp on average, with a spacer sequence of 30 bp and breakpoint junction of 6 bp. We showed that breakpoints of SITs preferentially occurred at nucleosome midpoints, aligned with Okazaki fragment termini. We further identified that SIT harbored in plasmids were precisely eliminated via DNA polymerase slippage over SIT‐derived hairpin structures in E. coli system. This study provides mechanistic insight into SIT origination and offers practical tools for future studies on genome rearrangements.