Most long-read DNA sequencing methods depend on library size selection techniques to controland/or improve read lengths. In Oxford Nanopore and PacBio sequencing protocols, sampleloading is dependent on diffusion of library molecules to the reader surface (waveguide surfacefor PacBio, or membrane for Oxford). Since small molecules have a higher rate of diffusion thanlarger molecules, size selection to eliminate small library fragments can greatly improve libraryread lengths. Popular methods for elimination of small library elements (generally <6-10kb) inlong read sequencing are preparative agarose gel electrophoresis (BluePippin, SageELF,PippinHT from Sage Science, Inc.), and PEG precipitation (Short Read Eliminator Kit,Circulomics, as well as home brew PEG methods). Of these methods, preparative gelelectrophoresis is superior in rejecting small DNA molecules, but requires specializedinstruments, consumable gel cassettes with bulky gel columns (6-10cm long) and low per-cassette sample throughputs, and long run times. In many long-read sequencing workflows,size selection is the most time-intensive step of the workflow.As long-read sequencing methods are rapidly becoming essential clinical research tools -- andincreasingly attracting attention of medical testing labs -- it is important to develop size selectionequipment and methods that have improved size range, improved resolution at large fragmentsizes (10kb-200kb), faster run times, and higher sample throughput per run cycle.The goal of the present application is develop new preparative gel electrophoresis systems thatutilize the nonlinear response of large DNAs: 1) while reorienting in response to certain pulsedfield conditions, or 2) when subjected to forward and reverse voltage pulses of differentstrength. In both cases, we envision size selection processes in which targeted size fractionsmove very little and are recovered within (or near) the sample loading position. If successful, gelsizes and run times can be decreased dramatically, thereby relieving a bottleneck in long-readworkflows that benefit from stringent size selection. Our ultimate goal is a family of sizeselection products that utilize SBS-format, automation-friendly gel cassettes with 48 or 96 wellsample capacity.
Public Health Relevance Statement: Project Narrative
Most long-read DNA sequencing methods depend on size selection techniques to control and/or
improve read lengths. However, in most long-read library preparation workflows, size selection
is the most time-intensive step. Since long-read sequencing is rapidly becoming an essential
clinical research tool -- and attracting increasing attention from medical testing labs -- it is
important to develop size selection equipment and methods that have improved size range,
improved resolution at large fragment sizes, faster run times, and higher sample throughput per
run cycle. This proposal seeks to develop a family of size selection products that utilize high-
capacity, fast running, automation-friendly gel cassettes with 48 or 96 sample capacity.
Project Terms:
