3rd generation sequencing technologies have revolutionized our understanding of the structure-function of the genome and the accuracy of reference assemblies. Transformative advances from Pacific Biosciences, Oxford Nanopore, 10X Genomics, and BioNano Genomics have created a resurgent need for high molecular weight (MW) DNA of the utmost quality and for new technologies to effectively process it. Library preparation for most long-read sequencing technologies requires size selection purification using AMPure beads to remove smaller background molecules (e.g. <200 bp) from the desired library products and separate pulsed field gel electrophoresis to then isolate the longest library products from shorter fragmented products (e.g. <6 kb). However, AMPure has low recovery efficiency (<25%) for high MW DNA, preferentially losing the longest, most desired DNA molecules while PFGE instruments such as Sage Science's BluePippin are very slow (8 hours) and also damage DNA during the long PFGE process, necessitating subsequent enzymatic repair. In this Phase I SBIR, we will develop 2 technologies for rapid size selection of both small and large DNA using our Nanobind silica nanomaterial to replace inefficient AMPure purifications and slow PFGE separations with a rapid bind, wash, and elute process. Nanobind is a novel thermoplastic nanomaterial that can be inexpensively manufactured and is capable of extracting higher quality DNA than any competing method. The low shear, non-porous substrate employs a novel tentacle binding mechanism that condenses DNA onto the surface of the disk and protects it from fragmentation and other damage to obtain high quality, high MW DNA. First, we will develop Nanobind Small DNA Size Select with tunable cutoffs between 50 - 400 bp to purify small molecules in enzymatic reactions with high recovery efficiency of high MW DNA. Second, we will integrate molecular crowding to create Nanobind Large DNA Size Select with tunable cutoffs between 200 bp - 10 kb to enhance read lengths and replace slow PFGE purifications. No magnetic particle or spin column technology is capable of size selection in the kilobase range. Finally, we will validate Nanobind Size Select by performing Pacific Biosciences SMRT sequencing and comparing against libraries made using the standard AMPure/BluePippin approaches.
For long-read sequencing to decrease in cost and to gain a foothold in clinical applications, robust and easy sample preparation technologies must be developed. The proposed project will develop a nanomaterial enabled process to reduce library preparation times and improve sequencing data quality and read lengths.
