Scaffolding of a bacterial genome using MinION nanopore sequencing

Abstract

Second generation sequencing has revolutionized genomic studies. However, most genomes contain repeated DNA elements that are longer than the read lengths achievable with typical sequencers, so the genomic order of several generated contigs cannot be easily resolved. A new generation of sequencers offering substantially longer reads is emerging, notably the Pacific Biosciences (PacBio) RS II system and the MinION system, released in early 2014 by Oxford Nanopore Technologies through an early access program. The latter has highly advantageous portability and sequences samples by measuring changes in ionic current when single-stranded DNA molecules are translocated through nanopores. We show that the MinION system produces long reads with high mapability that can be used for scaffolding bacterial genomes, despite currently producing substantially higher error rates than PacBio reads. With further development we anticipate that MinION will be useful not only for assembling genomes, but also for rapid detection of organisms, potentially in the field.

Figure 1. Quality of MinION (R7.3) and PacBio sequencing reads.

(a) Length distribution of the reads. MinION reads are divided into three length categories that are coloured separately. Note that the high number of MinION reads of about 3.5 kb originate from the ligation control fragment. (b) Mapability of PacBio and MinION reads divided into the same length categories as in (a). Read alignment length is the fraction of the reads covered in the BLAST alignment against the reference genome. (c) Mean frequencies of deletion, insertion and substitution errors per nucleotide per read for MinION sequence reads in four genomic regions: whole genome (32% GC), high GC-content regions (47.8% GC), A/T and G/C monomers at least 5 bp long. (d) Consensus accuracy versus average read coverage of the genome. Different coverages were obtained by subsampling the reads from a single MinION run.

Figure 2. Overview of the FSC996 reference genome and the MinION data.

The outermost track represents the reference genome with genomic positions (in kb). The blue lines provide a visual representation of every MinION read longer than 2 kb used in the analysis and its genomic alignment position (tile layers limited to 100). The black lines show the contigs generated from the assembly of Illumina Hiseq reads and their alignment positions. The inner circle shows a heat map of the GC content of the reference genome, with the brightest colour representing 20% GC and the darkest 40%.