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.
Highlights
Whole genome sequencing (WGS) reveals important genetic information of an organism that can be linked to specific phenotypes and enable genetic engineering approaches (Mardis 2008; Ng and Kirkness 2010)
To obtain a complete chromosome level de novo assembly of Saccharomyces cerevisiae CENPK113–7D, we performed long-read sequencing on the Oxford Nanopore Technology (ONT) MinION platform
In addition to the lesser fragmentation, the addition of 770 Kbp of previously unassembled sequence led to the identification and accurate placement of 284 additional ORFs spread out over the genome. These newly assembled genes showed overrepresentation for cell wall and cell periphery compartmentalisation and relate to functions such as sugar utilisation, amino acid uptake, metal ion metabolism, flocculation and tolerance to various stresses. While many of these genes were already present in the short-read assembly of CEN.PK113–7D, copy number was shown to be an important factor determining the adaptation of strains to specific growth conditions (Brown, Murray and Verstrepen 2010)
Summary
Whole genome sequencing (WGS) reveals important genetic information of an organism that can be linked to specific phenotypes and enable genetic engineering approaches (Mardis 2008; Ng and Kirkness 2010). The sequence reads obtained are relatively short: between 35 and 1000 bp (van Dijk et al 2014). This poses challenges as genomes have long stretches of repetitive sequences of several thousand nucleotides in length and can only be characterised if a read spans the repetitive region and has a unique fit to the flanking ends (Matheson, Parsons and Gammie 2017). The resulting assembly consists of dozens to hundreds of sequence fragments, commonly referred to as contigs. These contigs are either analysed independently or ordered and joined together adjacently based on their alignment to a closely related reference genome. Reference-based joining of contigs into so-called scaffolds is based on the assumption that the genetic structure of the sequenced strain is identical to that of the reference genome—potentially concealing existing genetic variation
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