Genome assembly using Nanopore-guided long and error-free DNA reads.

Mohammed-Amin Madoui,Arnaud Lemainque,Jean-Marc Aury,Patrick Wincker,Caroline Belser,Stefan Engelen,Adriana Alberti,Corinne Cruaud,Laurie Bertrand

doi:10.1186/s12864-015-1519-z

Abstract

BackgroundLong-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. Moreover by providing long-range information, it could also solve haplotype phasing. However, existing long-read technologies still have several limitations that complicate their use for most research laboratories, as well as in large and/or complex genome projects. In 2014, Oxford Nanopore released the MinION® device, a small and low-cost single-molecule nanopore sequencer, which offers the possibility of sequencing long DNA fragments.ResultsThe assembly of long reads generated using the Oxford Nanopore MinION® instrument is challenging as existing assemblers were not implemented to deal with long reads exhibiting close to 30% of errors. Here, we presented a hybrid approach developed to take advantage of data generated using MinION® device. We sequenced a well-known bacterium, Acinetobacter baylyi ADP1 and applied our method to obtain a highly contiguous (one single contig) and accurate genome assembly even in repetitive regions, in contrast to an Illumina-only assembly. Our hybrid strategy was able to generate NaS (Nanopore Synthetic-long) reads up to 60 kb that aligned entirely and with no error to the reference genome and that spanned highly conserved repetitive regions. The average accuracy of NaS reads reached 99.99% without losing the initial size of the input MinION® reads.ConclusionsWe described NaS tool, a hybrid approach allowing the sequencing of microbial genomes using the MinION® device. Our method, based ideally on 20x and 50x of NaS and Illumina reads respectively, provides an efficient and cost-effective way of sequencing microbial or small eukaryotic genomes in a very short time even in small facilities. Moreover, we demonstrated that although the Oxford Nanopore technology is a relatively new sequencing technology, currently with a high error rate, it is already useful in the generation of high-quality genome assemblies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1519-z) contains supplementary material, which is available to authorized users.

Highlights

Long-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes
As existing assembly softwares were not implemented to deal with long reads with a high error rate, we developed a method based on a combination of two sequencing technologies: Oxford Nanopore and Illumina, to produce long and accurate synthetic reads before assembly
As previously reported [8,9,10], we observed a low mappability on the reference genome [14]; 83.2% of 2D reads and 16.6% of 1D reads were aligned (Figure 1 and Table 2)

Summary

Introduction

Long-read sequencing technologies were launched a few years ago, and in contrast with short-read sequencing technologies, they offered a promise of solving assembly problems for large and complex genomes. The time needed for library construction may be a limitation in a time-constrained project, and again does not make it accessible to the whole research community This year, Oxford Nanopore Technologies Ltd released the MinION® device, a single-molecule nanopore sequencer connected to a laptop through a USB 3.0 interface, to hundreds of members of the MinION® Access Programme (MAP) who are testing the new device. The technology is based on an array of nanopores embedded on a chip that detects consecutive 5-mers of a single-strand DNA molecule by electrical sensing [7] This new technology provides several advantages: the MinION® device is small and low cost, the library construction involves a simplified method, no amplification step is needed, and data acquisition and analyses occur in real time. Otherwise only the forward strand sequence is provided (called 1D read)

Methods

Results

Conclusion