Novel target capture DNA library preparation method using CircLigase-mediated hook ligation

•A new target capture DNA library preparation method was developed.•A complementary sequence on single-strand oligos can improve CircLigase ligation efficiency.•Hook ligation uses CircLigase to achieve single-strand DNA ligation to a hook oligo containing a complementary sequence. Target capture DNA library preparation methods primarily use the biotin-streptavidin interaction to enrich target DNA, requiring complex operations and a time-consuming workflow with a series of wash steps at regulated temperatures. Here, a new method is presented termed ‘hook ligation’ for target DNA library preparation, using CircLigase to capture target DNA. The concept was validated by library preparation, sequencing, and acquisition of target DNA fragment information through bioinformatics analysis. An efficient reference point for single-strand DNA ligation to a hook sequence using CircLigase is also provided and it is shown that the efficiency can be influenced by the length and the position of the complementary area on the target DNA.