Yeast genomes can be assembled from sequencing data, but genome integrations and episomal plasmids often fail to be resolved with accuracy, completeness, and contiguity. Resolution of these features is critical for many synthetic biology applications, including strain quality control and identifying engineering in unknown samples. Here, we report an integrated workflow, named Prymetime, that uses sequencing reads from inexpensive NGS platforms, assembly and error correction software, and a list of synthetic biology parts to achieve accurate whole genome sequences of yeasts with engineering annotated. To build the workflow, we first determined which sequencing methods and software packages returned an accurate, complete, and contiguous genome of an engineered S. cerevisiae strain with two similar plasmids and an integrated pathway. We then developed a sequence feature annotation step that labels synthetic biology parts from a standard list of yeast engineering sequences or from a custom sequence list. We validated the workflow by sequencing a collection of 15 engineered yeasts built from different parent S. cerevisiae and nonconventional yeast strains. We show that each integrated pathway and episomal plasmid can be correctly assembled and annotated, even in strains that have part repeats and multiple similar plasmids. Interestingly, Prymetime was able to identify deletions and unintended integrations that were subsequently confirmed by other methods. Furthermore, the whole genomes are accurate, complete, and contiguous. To illustrate this clearly, we used a publicly available S. cerevisiae CEN.PK113 reference genome and the accompanying reads to show that a Prymetime genome assembly is equivalent to the reference using several standard metrics. Finally, we used Prymetime to resequence the nonconventional yeasts Y. lipolytica Po1f and K. phaffii CBS 7435, producing an improved genome assembly for each strain. Thus, our workflow can achieve accurate, complete, and contiguous whole genome sequences of yeast strains before and after engineering. Therefore, Prymetime enables NGS-based strain quality control through assembly and identification of engineering features.

中文翻译：

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纳米孔全基因组测序验证酵母中的基因工程

可以从测序数据中组装酵母基因组，但是基因组整合和附加型质粒通常无法准确，完整和连续地解析。这些特征的分辨率对于许多合成生物学应用至关重要，包括菌株质量控制和鉴定未知样品中的工程。在这里，我们报告了一个名为Prymetime的集成工作流，该工作流使用了来自廉价NGS平台的测序读取，组装和错误校正软件以及一系列合成生物学部分，以实现带有工程注释的准确的酵母全基因组序列。为了构建工作流程，我们首先确定哪些测序方法和软件包返回了工程酿酒酵母的准确，完整和连续的基因组。具有两个相似质粒和整合途径的菌株。然后，我们开发了序列特征注释步骤，该步骤从酵母工程序列的标准列表或自定义序列列表中标记合成生物学部分。我们通过对从不同的酿酒酵母和非常规酵母菌株构建的15种工程酵母进行测序来验证工作流程。我们显示，即使在具有部分重复和多个相似质粒的菌株中，每个整合的途径和附加型质粒也可以正确组装和注释。有趣的是，Prymetime能够识别随后被其他方法确认的缺失和意外整合。此外，整个基因组是准确，完整和连续的。为了清楚地说明这一点，我们使用了公开发布的酿酒酵母CEN.PK113参考基因组和随附的读物显示，使用多个标准指标，Prymetime基因组装配等同于参考。最后，我们使用Prymetime对非常规酵母解脂耶氏酵母Po1f和K. phaffii CBS 7435进行了重新测序，从而为每个菌株产生了改良的基因组装配。因此，我们的工作流程可以在工程改造前后实现准确，完整和连续的酵母菌株全基因组序列。因此，Prymetime通过组装和识别工程特征实现基于NGS的应变质量控制。