The homokaryotic Coprinopsis cinerea strain A43mut B43mut pab1-1 #326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factors A and B result in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with the C. cinerea strain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein (RasGAP) was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform.

的同核Coprinopsis孢菌株A43mut B43mut pab1-1＃326是用于在蘑菇形成真菌发育研究一种广泛使用的实验模型。它可以在特定的人工培养基上生长，并在两周内完成整个生命周期。交配型因子A和B的突变导致夹子形成和结果的特殊功能，无需交配。此功能允许在同核遗传背景下进行调查和操作。当前菌株#326 的基因组组装基于短读长测序数据并且高度碎片化，导致基因注释和下游分析存在偏差。在这里，我们报告了菌株 #326 的染色体水平基因组组装。Oxford Nanopore Technology (ONT) MinION 测序用于获得长读数。使用 Illumina 短读长来完善序列。组合组装产生 13 条染色体和线粒体基因组作为单独的支架。该程序集具有 15,250 个注释基因，与C. cinerea具有高度同线性菌株 Okayama-7 #130。这个程序集在连续性和注释上有很大的改进。它是进一步基因组研究的合适参考，尤其是 ONT 长读长中的遗传、基因组和转录组分析。可以鉴定和验证六个诱变和顺铂筛选的突变体中的单核苷酸变异体和结构变异体。Ras GTPase 激活蛋白 ( RasGAP) 中的66 bp 缺失) 在所有突变体中都有发现。为了更好地利用ONT测序平台，我们修改了基于磁珠的丝状真菌高分子量基因组DNA分离方案。该研究展示了使用 MinION 构建真菌参考基因组并在单个实验室进行下游研究。提出了一个实验工作流程，从 DNA 分离和全基因组测序，到基因组组装和变异识别。我们的结果为 MinION 测序平台上的真菌基因组分析提供了解决方案和参数。