Mate-pair Library Construction with Controlled Polymerization Enables Comprehensive Structural Rearrangement Detection

It is important, yet difficult, to identify genomic structural rearrangements associated with congenital diseases or tumors. Mate-pair sequencing enables the positioning of a long DNA fragment with complete and precise breakpoints and has therefore become a common diagnostic approach for identifying chromosomal aberrations. Several methods are currently used for detection. However, due to cost, the need for large input quantity, and operation complexity, existing workflows are unsuitable for large-scale clinical studies. Herein, we describe a new process that couples advanced controlled polymerization with a non-conventional adapter ligation to generate mate-pairs with desirable length that yield minimal GC bias and improved coverage uniformity. Compared to other methods, our strategy can achieve 8-fold improved DNA circularization efficiency, a 39.3-fold reduction of read-pairs that do not cross the circularization junction, and the lowest chimeric rate, collectively producing an ∼50% increase of physical coverage. In a proof-of-concept study using five insertion translocations, the structural rearrangements were comprehensively detected using longer 100-bp reads enabled by this approach. Based on its ability to identify single-nucleotide-resolution changes, this approach shows promise as an integrated method for the comprehensive detection of genomic variants at a fraction of current cost.