We proposed to develop Nanobind reaction purification and size selection methods for long-read sequencing library preparation. These methods use our Nanobind magnetic disks to purify enzymatic reactions and to enhance read lengths by removing short DNA during long-read sequencing library preparation. Nanobind disks are small 1-5 mm diameter magnetic disks covered with a high density of micro- and nanostructured silica. Compared to magnetic particles, Nanobind disks are extraordinarily gentle, preventing DNA shearing and enabling high efficiency recovery of DNA surpassing megabase in length. We currently sell DNA extraction kits that enable isolation of ultra high molecular weight (UHMW) megabase-sized DNA from a wide variety of sample types. To evaluate how gentle the Nanobind reaction purifications are, we tested them in an ultra-long sequencing protocol capable of generating 100 kb ? 1+ Mb length reads. This method is capable of generating extremely high read length N50 in the 100 ? 200 kb range and 10 times more megabase reads than previous ultra long methods. This supplement will allow additional development and sequencing tests on the Nanobind ultra long sequencing protocols. Successful validation of the method on PromethION will lead to a significant enhancement in data throughput and corresponding in reduction of cost, time, and manpower, enabling an immediate impact via translation into projects such as the NHGRI pan-genome project and a long-term impact by dramatically reducing the cost of ultra long sequencing.
Ultra long sequencing methods are capable of generating continuous reads spanning 100 kb ? 1+ Mb in length. These ultra long reads can be used to create improved reference genomes that expand our understanding of genome structure and function and illuminate dark regions of the genome. This can help identify of genetic underpinnings of disease, therapeutic targets, and new diagnostic tests.
