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

7. Project summary/Abstract
Recent advances by the two major long-read sequencing companies, PacBio and Oxford Nanopore (ONT),
have enabled complete sequencing of the human genome. As a result, many major genomic research
programs (NIH’s All of Us, T2T consortium, Human Pangenome Reference Consortium) as well as many
genetic testing companies are racing to implement long-read whole genome sequencing (WGS) as a standard
of care for genetic testing in the near term, and for comprehensive precision medicine programs of the future.
For such an effort to succeed, long read sequencing workflows must be adapted to use high-throughput
automated equipment to reduce overall costs, to improve workflow reproducibility, and to eliminate sources of
operator error. In both long-read technologies, library loading onto the read positions in the flow cell (zero-
mode waveguides in PacBio, pores in ONT) is diffusion-limited, and smaller library elements will be over-
represented in the sequencing output relative to the contribution of longer, more informative library elements.
For this reason, size-selection to eliminate shorter library molecules is widely practiced by users of both
sequencing platforms. Size-selection methods that have been used in long-read library preparation include
size-selective precipitation, customized Ampure bead isolation methods, and automated preparative agarose
gel electrophoresis. Size-selection by preparative electrophoresis is far more effective and flexible than
precipitation or magnetic bead methods, but it suffers from incompatibility with conventional liquid handling
instrumentation. This incompatibility is a challenge for implementing long-read sequencing broadly in biological
research and genetic testing. This proposal seeks to develop a high-throughput, cost-effective gel-based size-
selection system that is easy to integrate with standard liquid handling robotics. The system is designed to be
flexible in physical configuration (capable of operation on or off the deck of a conventional liquid handling
robot), and flexible in size-selection performance, in order to serve size-selection needs of both long-read
platforms. Widespread availability of affordable long-read WGS will have enormous benefits for diagnosis of
genetic disease, but will also impact the development of new therapeutics as clinical sequencing programs
such as the US NIH All of Us program and the UK NHS Genomic Medicine Service generate large sequence
databases with linked patient medical records so that new therapeutic mechanisms and targets can be
identified. The proposed gel-plate system may also have applications in liquid biopsy assays. For instance, it
has been demonstrated that carefully size-selected plasma DNA fractions (smaller than ~150bp) can be
several-fold enriched in tumor or fetal DNA. The proposed gel-plate system could easily be adapted for such
liquid biopsy applications by increasing the agarose gel concentrations and changing the electrophoresis
voltage and time.

Grant Number: 5R44HG012531-03
NIH Institute/Center: NIH
Principal Investigator: TRUETT BOLES