High contiguityArabidopsis thalianagenome assembly with a single nanopore flow cell

Abstract

While many evolutionary questions can be answered by short read re-sequencing, presence/absence polymorphisms of genes and/or transposons have been largely ignored in large-scale intraspecific evolutionary studies. To enable the rigorous analysis of such variants, multiple high quality and contiguous genome assemblies are essential. Similarly, while genome assemblies based on short reads have made genomics accessible for non-reference species, these assemblies have limitations due to low contiguity. Long-read sequencers and long-read technologies have ushered in a new era of genome sequencing where the lengths of reads exceed those of most repeats. However, because these technologies are not only costly, but also time and compute intensive, it has been unclear how scalable they are. Here we demonstrate a fast and cost effective reference assembly for an Arabidopsis thaliana accession using the USB-sized Oxford Nanopore MinION sequencer and typical consumer computing hardware (4 Cores, 16Gb RAM). We assemble the accession KBS-Mac-74 into 62 contigs with an N50 length of 12.3 Mb covering 100% (119 Mb) of the non-repetitive genome. We demonstrate that the polished KBS-Mac-74 assembly is highly contiguous with BioNano optical genome maps, and of high per-base quality against a likewise polished Pacific Biosciences long-read assembly. The approach we implemented took a total of four days at a cost of less than 1,000 USD for sequencing consumables including instrument depreciation.

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

“…However, even when using multiple rounds of Racon and combining with nanopolish, the error rates remained higher, whereas gene completeness remained lower than when polished with Illumina data. This result is in line with recent data from the model plant Arabidopsis, where even after three rounds of Racon polishing, an additional round of Illumina data decreased error rates drastically (Michael et al, 2017). Thus, at the current stage, one would still recommend including Illumina data for polishing errors.…”

Recent advances in conservation and population genomics data analysis

“…Oxford Nanopore (https://nanoporetech.com/) and minION also use a single molecule approach to nucleotide identification that passes an ionic charge through a nanoscale hole and measures the changes in current as each molecule passes through (see Michael et al. (2017) for assembly comparison). SLR‐seq technologies, such as 10× Genomics (https://www.10xgenomics.com/) or Dovetail Genomics (https://dovetailgenomics.com/), still rely on short read technology and, using statistical phasing algorithms, have the capacity to assemble continuous haplotypes and scaffolds that can span whole chromosomes with high accuracy.…”

Is It Ordered Correctly? Validating Genome Assemblies by Optical Mapping

“…Michael et al (2017) describe the assembly of an entire Arabidopsis genome using Oxford Nanopore technology and confirm the assembly with Bionano optical mapping. PacBio frequently has been used to assemble the genomes of model species, and it combines well with optical mapping (Jiao et al, 2017), but sufficient coverage can be cost-limiting for many projects.…”

“…The sequence assembly phase includes using all of the available sequence data from short reads, mate pairs, and long reads to create contigs and scaffolds. Long-read single molecule sequencing technologies have made it possible to dramatically extend the length of sequence contigs, often including large portions of entire chromosomes (Michael et al, 2017). The genome assembly phase includes the integration of additional information such as prior assemblies, genetic maps, or Hi-C read pairs to order and orient the contigs into "pseudomolecules" that are representations of the chromosomes.…”