# High-throughput size-selection system for long-read sequencing library preparation

> **NIH NIH R43** · SAGE SCIENCE, INC. · 2022 · $320,862

## Abstract

Project Summary/Abstract
Most long-read DNA sequencing methods depend on library size selection techniques to control
and/or improve read lengths. In Oxford Nanopore and PacBio sequencing protocols, sample
loading is dependent on diffusion of library molecules to the reader surface (waveguide surface
for PacBio, or membrane for Oxford). Since small molecules have a higher rate of diffusion than
larger molecules, size selection to eliminate small library fragments can greatly improve library
read lengths. Popular methods for elimination of small library elements (generally <6-10kb) in
long 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 gel
electrophoresis is superior in rejecting small DNA molecules, but requires specialized
instruments, 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 -- and
increasingly attracting attention of medical testing labs -- it is important to develop size selection
equipment and methods that have improved size range, improved resolution at large fragment
sizes (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 that
utilize the nonlinear response of large DNAs: 1) while reorienting in response to certain pulsed
field conditions, or 2) when subjected to forward and reverse voltage pulses of different
strength. In both cases, we envision size selection processes in which targeted size fractions
move very little and are recovered within (or near) the sample loading position. If successful, gel
sizes and run times can be decreased dramatically, thereby relieving a bottleneck in long-read
workflows that benefit from stringent size selection. Our ultimate goal is a family of size
selection products that utilize SBS-format, automation-friendly gel cassettes with 48 or 96 well
sample capacity.

## Key facts

- **NIH application ID:** 10480521
- **Project number:** 1R43HG012531-01
- **Recipient organization:** SAGE SCIENCE, INC.
- **Principal Investigator:** TRUETT C BOLES
- **Activity code:** R43 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $320,862
- **Award type:** 1
- **Project period:** 2022-08-03 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10480521

## Citation

> US National Institutes of Health, RePORTER application 10480521, High-throughput size-selection system for long-read sequencing library preparation (1R43HG012531-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10480521. Licensed CC0.

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