Sequence assembly of large and repeat-rich plant genomes has been challenging, requiring substantial computational resources and often several complementary sequence assembly and genome mapping approaches. The recent development of fast and accurate long-read sequencing by circular consensus sequencing (CCS) on the PacBio platform may greatly increase the scope of plant pan-genome projects. Here, we compare current long-read sequencing platforms regarding their ability to rapidly generate contiguous sequence assemblies in pan-genome studies of barley (Hordeum vulgare). Most long-read assemblies are clearly superior to the current barley reference sequence based on short-reads. Assemblies derived from accurate long reads excel in most metrics, but the CCS approach was the most cost-effective strategy for assembling tens of barley genomes. A downsampling analysis indicated that 20-fold CCS coverage can yield very good sequence assemblies, while even five-fold CCS data may capture the complete sequence of most genes. We present an updated reference genome assembly for barley with near-complete representation of the repeat-rich intergenic space. Long-read assembly can underpin the construction of accurate and complete sequences of multiple genomes of a species to build pan-genome infrastructures in Triticeae crops and their wild relatives.

Center for Integrated Breeding Research Georg August University Göttingen Göttingen 37073 Germany

Department of Plant and Microbial Biology University of Zürich Zürich 8008 Switzerland

Department of Plant Sciences Crop Development Centre University of Saskatchewan Saskatoon SK S7N 5A8 Canada

German Centre for Integrative Biodiversity Research Halle Jena Leipzig Leipzig 04103 Germany

Global Institute for Food Security University of Saskatchewan Saskatoon SK S7N 4L8 Canada

HudsonAlpha Institute for Biotechnology Huntsville AL 35806

Institute of Experimental Botany of the Czech Academy of Sciences Centre of the Region Haná for Biotechnological and Agricultural Research Olomouc 78371 Czech Republic

Leibniz Institute of Plant Genetics and Crop Plant Research Gatersleben Seeland 06466 Germany

PGSB Plant Genome and Systems Biology Helmholtz Center Munich German Research Center for Environmental Health Neuherberg 85764 Germany

The Sainsbury Laboratory University of East Anglia Norwich NR4 7UH UK

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