De Novo Assembly of a New Solanum pennellii Accession Using Nanopore Sequencing

Abstract

Updates in nanopore technology have made it possible to obtain gigabases of sequence data. Prior to this, nanopore sequencing technology was mainly used to analyze microbial samples. Here, we describe the generation of a comprehensive nanopore sequencing data set with a median read length of 11,979 bp for a self-compatible accession of the wild tomato species Solanum pennellii We describe the assembly of its genome to a contig N50 of 2.5 MB. The assembly pipeline comprised initial read correction with Canu and assembly with SMARTdenovo. The resulting raw nanopore-based de novo genome is structurally highly similar to that of the reference S. pennellii LA716 accession but has a high error rate and was rich in homopolymer deletions. After polishing the assembly with Illumina reads, we obtained an error rate of <0.02% when assessed versus the same Illumina data. We obtained a gene completeness of 96.53%, slightly surpassing that of the reference S. pennellii Taken together, our data indicate that such long read sequencing data can be used to affordably sequence and assemble gigabase-sized plant genomes.

Figure 1.

Characteristics of the S. pennellii Genome and Its Assembly. (A) Circos visualization of variant distribution between S. pennellii LYC1722 and S. pennellii LA716. Distribution of single nucleotide polymorphisms (outer layer) and InDels (middle layer) is compared with the gene density (inner layer) for each chromosome of S. pennellii LA716 based on generated Illumina data for S. pennellii LYC1722. (B) The effect of randomly downsampling pass reads on the N50 produced by different assemblers. (C) Discrepancies between the assembly and the Illumina data over several rounds of Pilon correction. Dotted lines approximate expected discrepancy rates if Illumina data were mapped to a perfect reference.

Figure 2.

Violin Plots of Read Length per Library for Three Different Size-Selection Protocols. Read length distribution is shown for all 16 S. pennellii MinION libraries and the corresponding pass (blue) and failed (red) classified reads. Libraries are grouped by size selection protocol: (A) 15-kb cutoff, (B) 12 kb cutoff, and (C) 0.4x bead size selection. Filled dots indicate mean read length.

Figure 3.

The 6-mer Counts in the Polished Assembly versus Those in the Raw Reads. The 6-mers were counted both in the polished assembly and in the raw reads. Each 6-mer represents counts to both itself and to its reverse complement, i.e., AAAAAA represents both AAAAAA and TTTTTT. Red indicates the new Albacore basecaller, whereas blue and gray dots represent the raw and Canu-corrected Metrichor data. In each case, a trend line is added.