Resolving genetic engineering signatures in yeast on-site with the MinION and iSeq
July 14-18, 2019
Generating an assembly that captures all of the genome and plasmid modifications resulting from metabolic engineering is essential for quality control, connecting genotype to phenotype, establishing and protecting intellectual property, and generating “ground truth” for monitoring potential release events. Furthermore, high quality de novo assemblies can be used to accurately determine the presence and function of metabolic engineering signatures in an unknown sample. Here, we use two new inexpensive sequencers, the Oxford Nanopore MinION and the Illumina iSeq, to enable fast acquisition of sequence on-site. We also use new processing algorithms to enable fast transformation of sequence information into a high-quality genome assembly. The resulting pipeline can generate de novo microbial genome assemblies that capture complete chromosomal pathways and episomal plasmids. We further extend the pipeline to resequence six nonconventional yeasts of interest for metabolic engineering. To establish the most accurate workflow, we evaluated four nanopore de novo assemblers and three polishing algorithms at varying genome coverage depths for the lab strain S. cerevisiae CEN.PK113-7D. Our results show that (1) nanopore genome coverage depth must be at least 40X, (2) Flye and Canu are currently the best assemblers due to their combination of structure, completeness, and accuracy, and (3) Illumina data is essential for polishing. Our final pipeline (Figure 1A) generated a better S. cerevisiae CEN.PK113-7D assembly than the publicly available reference genome.
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Eric M. Young and Joseph H. Collins, "Resolving genetic engineering signatures in yeast on-site with the MinION and iSeq" in "Biochemical and Molecular Engineering XXI", Christina Chan, Michigan State University, USA Mattheos Koffas, RPI, USA Steffen Schaffer, Evonik Industries, Germany Rashmi Kshirsagar, Biogen, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/biochem_xxi/3