Ultralong Oxford Nanopore Reads Enable the Development of a Reference-Grade Perennial Ryegrass Genome Assembly.

Daniel Frei,Kentaro K Shimizu,Rie Shimizu-Inatsugi,Bruno Studer,Aki Morishima,Ingrid Stoffel-Studer,Jürg E Frey,Steven Yates,Daniel Grogg,Elisabeth Veekman,Dario Copetti,Laura Katz

doi:10.1093/gbe/evab159

Abstract

Despite the progress made in DNA sequencing over the last decade, reconstructing telomere-to-telomere genome assemblies of large and repeat-rich eukaryotic genomes is still difficult. More accurate basecalls or longer reads could address this issue, but no current sequencing platform can provide both simultaneously. Perennial ryegrass (Lolium perenne L.) is an example of an important species for which the lack of a reference genome assembly hindered a swift adoption of genomics-based methods into breeding programs. To fill this gap, we optimized the Oxford Nanopore Technologies’ sequencing protocol, obtaining sequencing reads with an N50 of 62 kb—a very high value for a plant sample. The assembly of such reads produced a highly complete (2.3 of 2.7 Gb), correct (QV 45), and contiguous (contig N50 and N90 11.74 and 3.34 Mb, respectively) genome assembly. We show how read length was key in determining the assembly contiguity. Sequence annotation revealed the dominance of transposable elements and repeated sequences (81.6% of the assembly) and identified 38,868 protein coding genes. Almost 90% of the bases could be anchored to seven pseudomolecules, providing the first high-quality haploid reference assembly for perennial ryegrass. This protocol will enable producing longer Oxford Nanopore Technology reads for more plant samples and ushering forage grasses into modern genomics-assisted breeding programs.

Highlights

Over the last three years, the sequencing performance of the Oxford Nanopore Technologies (ONT)platform has dramatically increased in terms of sequencing yield, accuracy, and read length ((Shafin et al.2020), https://bit.ly/3peI7xH, last accessed May 2021)
Perennial ryegrass (Lolium perenne L.) is an example of an important species for which the lack of a reference genome assembly hindered a swift adoption of genomics-based methods into breeding programs
We show how read length was key in determining the assembly contiguity

Summary

Significance statement

Sequencing eukaryotic genomes with long-read sequencing platforms is allowing to obtain genome assemblies of unprecedented quality for many non-model organisms. Especially in genomes with a high amount of long repeats, completeness and contiguity are limited by the quality (accuracy and/or length) of the input data. We present an innovative protocol for Oxford Nanopore. Technologies’ genomic plant DNA library preparation that considerably increases read length. We show how these exceptionally longer reads were key in obtaining a perennial ryegrass genome assembly with unprecedented statistics, both within its genus and among other plants of similar complexity. This work makes available a highly complete and contiguous genome assembly and the laboratory protocol necessary to produce long read data

Introduction

Results and Discussion

Materials and methods

Literature cited