Abstract
Yeast whole genome sequencing (WGS) lacks end-to-end workflows that identify genetic engineering. Here we present Prymetime, a tool that assembles yeast plasmids and chromosomes and annotates genetic engineering sequences. It is a hybrid workflow—it uses short and long reads as inputs to perform separate linear and circular assembly steps. This structure is necessary to accurately resolve genetic engineering sequences in plasmids and the genome. We show this by assembling diverse engineered yeasts, in some cases revealing unintended deletions and integrations. Furthermore, the resulting whole genomes are high quality, although the underlying assembly software does not consistently resolve highly repetitive genome features. Finally, we assemble plasmids and genome integrations from metagenomic sequencing, even with 1 engineered cell in 1000. This work is a blueprint for building WGS workflows and establishes WGS-based identification of yeast genetic engineering.
Highlights
Yeast whole genome sequencing (WGS) lacks end-to-end workflows that identify genetic engineering
We set a standard that a genome assembly workflow must be able to resolve chromosomal integrations and multiple plasmids used in yeast engineering
We developed a gentle genomic DNA isolation protocol which increased average nanopore read length and reduced variance (Supplementary Figure S1)
Summary
Yeast whole genome sequencing (WGS) lacks end-to-end workflows that identify genetic engineering. It is a hybrid workflow—it uses short and long reads as inputs to perform separate linear and circular assembly steps This structure is necessary to accurately resolve genetic engineering sequences in plasmids and the genome. Both hybrid and long read with error correction assembly approaches currently hold the most promise to achieve accurate genome sequence and structure at low read depths, primarily because two independent technologies validate basecalls This entails the use of two sequencing technologies, thereby increasing costs and time. The resulting workflow is named Prymetime, "Pipeline for Recombinant Yeast genoMEs That Identifies Markers of Engineering." Through a variety of demonstrations, we show that Prymetime can validate genetic engineering, produce high quality whole genome sequences, and detect engineering in metagenomic samples This tool is broadly useful for strain validation, release monitoring, protecting intellectual property, and investigating engineering in unknown samples
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