High contiguity Arabidopsis thaliana genome assembly with a single nanopore flow cell

Todd P Michael,Felix Bemm,S Timothy Motley,Christa Lanz,Detlef Weigel,Olivier Loudet,Justin P Sandoval,Joseph R Ecker,Florian Jupe

doi:10.1038/s41467-018-03016-2

Abstract

The handheld Oxford Nanopore MinION sequencer generates ultra-long reads with minimal cost and time requirements, which makes sequencing genomes at the bench feasible. Here, we sequence the gold standard Arabidopsis thaliana genome (KBS-Mac-74 accession) on the bench with the MinION sequencer, and assemble the genome using typical consumer computing hardware (4 Cores, 16 Gb RAM) into chromosome arms (62 contigs with an N50 length of 12.3 Mb). We validate the contiguity and quality of the assembly with two independent single-molecule technologies, Bionano optical genome maps and Pacific Biosciences Sequel sequencing. The new A. thaliana KBS-Mac-74 genome enables resolution of a quantitative trait locus that had previously been recalcitrant to a Sanger-based BAC sequencing approach. In summary, we demonstrate that even when the purpose is to understand complex structural variation at a single region of the genome, complete genome assembly is becoming the simplest way to achieve this goal.

Highlights

The handheld Oxford Nanopore MinION sequencer generates ultra-long reads with minimal cost and time requirements, which makes sequencing genomes at the bench feasible
We show how one can rapidly and inexpensively resolve structural variation (SV) at a quantitative trait locus (QTL) in A. thaliana by taking advantage of a highly contiguous genome assembly produced from 2 μg genomic DNA on just a single Oxford Nanopore Technology (ONT) MinION flow cell (R9.4)
The final assembly, which was generated on a laptop in only 4 days, covers 100% of the nonrepetitive genome space, with only a fraction of the gaps present in the current gold standard Arabidopsis TAIR10 assembly

Summary

Introduction

The handheld Oxford Nanopore MinION sequencer generates ultra-long reads with minimal cost and time requirements, which makes sequencing genomes at the bench feasible. The read lengths of third-generation sequencing technologies, such as SMRT Sequencing from Pacific Biosciences (PacBio), exceed those of major repeats in many genomes, and are enabling highly contiguous genome assemblies[10,11]. Repetitive regions such as the ribosomal DNA (rDNA) and centromeres, still remain mostly unassembled with thirdgeneration technologies due to limitations in read length and error rate. We show how one can rapidly and inexpensively resolve structural variation (SV) at a quantitative trait locus (QTL) in A. thaliana by taking advantage of a highly contiguous genome assembly produced from 2 μg genomic DNA on just a single ONT MinION flow cell (R9.4). We show the utility of such a rapidly created genome by identifying an intrachromosomal duplication responsible for a growth phenotype that could not be resolved with a Sanger-based BAC sequencing approach

Methods

Results

Conclusion