Ryan Lorig-Roach1, Melissa Meredith1, Jean Monlong1, Miten Jain2, Hugh E. Olsen1, Brandy McNulty1, David Porubsky3, Tessa G. Montague4,5, Julian K. Lucas1, Chris Condon1, Jordan M. Eizenga1, Sissel Juul6, Sean K. McKenzie6, Sara E. Simmonds7, Jimin Park1, Mobin Asri1, Sergey Koren8, Evan E. Eichler9,10, Richard Axel4,5, Bruce Martin7, Paolo Carnevali7, Karen H. Miga1 and Benedict Paten1 1UC Santa Cruz Genomics Institute, University of California, Santa Cruz, Santa Cruz, California 95060, USA; 2Department of Bioengineering, Department of Physics, Northeastern University, Boston, Massachusetts 02120, USA; 3Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington 98195, USA; 4The Mortimer B. Zuckerman Mind Brain Behavior Institute, Department of Neuroscience, Columbia University, New York, New York 10027, USA; 5Howard Hughes Medical Institute, Columbia University, New York, New York 10032, USA; 6Oxford Nanopore Technologies Incorporated, New York, New York 10013, USA; 7Chan Zuckerberg Initiative Foundation, Redwood City, California 94063, USA; 8Genome Informatics Section, Computational and Statistical Genomics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20894, USA; 9Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington 98195, USA; 10Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA Corresponding authors: rlorigroucsc.edu, pacarnevucsc.edu, bpatenucsc.edu Abstract

Reference-free genome phasing is vital for understanding allele inheritance and the impact of single-molecule DNA variation on phenotypes. To achieve thorough phasing across homozygous or repetitive regions of the genome, long-read sequencing technologies are often used to perform phased de novo assembly. As a step toward reducing the cost and complexity of this type of analysis, we describe new methods for accurately phasing Oxford Nanopore Technologies (ONT) sequence data with the Shasta genome assembler and a modular tool for extending phasing to the chromosome scale called GFAse. We test using new variants of ONT PromethION sequencing, including those using proximity ligation, and show that newer, higher accuracy ONT reads substantially improve assembly quality.

Received July 19, 2023. Accepted March 19, 2024.