Nanopore sequencing enables near-completede novoassembly ofSaccharomyces cerevisiaereference strain CEN.PK113-7D

Abstract

The haploid Saccharomyces cerevisiae strain CEN.PK113-7D is a popular model system for metabolic engineering and systems biology research. Current genome assemblies are based on short-read sequencing data scaffolded based on homology to strain S288C. However, these assemblies contain large sequence gaps, particularly in subtelomeric regions, and the assumption of perfect homology to S288C for scaffolding introduces bias. In this study, we obtained a near-complete genome assembly of CEN.PK113-7D using only Oxford Nanopore Technology's MinION sequencing platform. Fifteen of the 16 chromosomes, the mitochondrial genome and the 2-μm plasmid are assembled in single contigs and all but one chromosome starts or ends in a telomere repeat. This improved genome assembly contains 770 Kbp of added sequence containing 248 gene annotations in comparison to the previous assembly of CEN.PK113-7D. Many of these genes encode functions determining fitness in specific growth conditions and are therefore highly relevant for various industrial applications. Furthermore, we discovered a translocation between chromosomes III and VIII that caused misidentification of a MAL locus in the previous CEN.PK113-7D assembly. This study demonstrates the power of long-read sequencing by providing a high-quality reference assembly and annotation of CEN.PK113-7D and places a caveat on assumed genome stability of microorganisms.

Variants in RNA-Seq data show a continued mutation rate during strain preservation ofSchizophyllum commune

“…Hence, the absence of ku80 might not have an impact on single nucleotide polymorphisms, but rather induce structural variations such as indels or inversions. Our method to detect variations of DNA from RNA sequence data was not designed to detect these larger variations and the use of short-reads for RNA-seq precludes the study of large indels and translocations that long reads would ameliorate (Salazar et al, 2017). For the knock-out strains that are derived from the ku80 strain, we observed a lower mutation rate.…”

GENOME REPORT: High-quality genome assemblies of 15 Drosophila species generated using Nanopore sequencing

“…A highly contiguous genome may in fact contain many errors that make it difficult to use for downstream analysis such as gene finding or SNP calling. We did not expect our Nanopore-only assemblies to be of high quality because raw Nanopore reads have an observed error rate of 10-15% (Li 2016;Simpson et al 2017;Salazar et al 2017). It is, however, possible to improve assembly quality through one or more rounds of polishing, which is a process that improves assembly quality using the original sequencing reads, reads from another technology with a relatively low error rate (e.g., Illumina data), or a combination of both.…”

“…Genome assemblies using Oxford Nanopore sequencing technology have been reported for several species, including Saccharomyces cerevisiae (Salazar et al 2017), Arabidopsis thaliana (Michael et al 2017), Caenorhabditis elegans (Tyson et al 2017), and Homo sapiens (Jain et al 2018). This technology measures changes in current as a molecule (currently either DNA or RNA) passes through a small pore in a membrane.…”

Alpaca: a kmer-based approach for investigating mosaic structures in microbial genomes

“…We are currently at a crossroads of microbial genome sequencing data-types: single-molecule sequencing technologies (such as PacBio and Oxford Nanopore) are enabling complete characterization of (individual) microbial genomes (Loman et al, 2015;Rodríguez et al, 2015;Salazar et al, 2017;Yue et al, 2017), while past and current short-read sequencing projects of hundreds to thousands of microbial strains contain vast information about their population diversity (Manson et al, 2017;Peter et al, 2018). Combining both genomic data-types can thus aid in our understanding of the potential mosaic structures in microbial populations.…”