Gueidan Cécile, Li Lan
Australian National Herbarium, National Research Collections Australia, NCMI, CSIRO, Canberra, ACT, 2601, Australia Australian National Herbarium, National Research Collections Australia Canberra Australia.
MycoKeys. 2022 Feb 2;86:195-212. doi: 10.3897/mycokeys.86.77431. eCollection 2022.
Reference sequence databases are critical to the accurate detection and identification of fungi in the environment. As repositories of large numbers of well-curated specimens, herbaria and fungal culture collections have the material resources to generate sequence data for large number of taxa, and could therefore allow filling taxonomic gaps often present in reference sequence databases. Financial resources to do that are however often lacking, so that recent efforts have focused on decreasing sequencing cost by increasing the number of multiplexed samples per sequencing run while maintaining high sequence quality. Following a previous study that aimed at decreasing sequencing cost for lichen specimens by generating fungal ITS barcodes for 96 specimens using PacBio amplicon sequencing, we present a method that further decreases lichen specimen metabarcoding costs. A total of 384 mixed DNA extracts obtained from lichen herbarium specimens, mostly from the four genera , , and , were used to generate new fungal ITS sequences using a Sequel I sequencing platform and the PacBio M13 barcoded primers. The average success rate across all taxa was high (86.5%), with particularly high rates for the crustose saxicolous taxa (, and others; 93.3%) and the terricolous squamulose taxa ( and others; 96.5%). On the other hand, the success rate for the foliose genus was lower (60.4%). With this taxon sampling, greater specimen age did not appear to impact sequencing success. In fact, the 1966-1980 collection date category showed the highest success rate (97.3%). Compared to the previous study, the abundance-based sequence denoising method showed some limitations, but the cost of generating ITS barcodes was further decreased thanks to the higher multiplexing level. In addition to contributing new ITS barcodes for specimens of four interesting lichen genera, this study further highlights the potential and challenges of using new sequencing technologies on collection specimens to generate DNA sequences for reference databases.
参考序列数据库对于准确检测和鉴定环境中的真菌至关重要。作为大量精心整理标本的储存库，植物标本馆和真菌培养物保藏中心拥有为大量分类单元生成序列数据的物质资源，因此可以填补参考序列数据库中经常存在的分类学空白。然而，进行此项工作的资金资源往往匮乏，因此近期的努力集中在通过增加每次测序运行中多重样本的数量来降低测序成本，同时保持高序列质量。继之前一项旨在通过使用PacBio扩增子测序为96个地衣标本生成真菌ITS条形码来降低地衣标本测序成本的研究之后，我们提出了一种进一步降低地衣标本宏条形码测序成本的方法。总共384份从地衣植物标本馆标本中提取的混合DNA提取物（主要来自四个属， 、 、 和 ），被用于使用Sequel I测序平台和PacBio M13条形码引物生成新的真菌ITS序列。所有分类单元的平均成功率很高（86.5%），其中壳状石生分类单元（ 、 及其他；93.3%）和土生鳞叶状分类单元（ 及其他；96.5%）的成功率尤其高。另一方面，叶状属 的成功率较低（60.4%）。通过这种分类单元抽样，标本年代越久似乎并未影响测序成功率。事实上，1966 - 1980年收集日期类别的成功率最高（97.3%）。与之前的研究相比，基于丰度的序列去噪方法显示出一些局限性，但由于更高的多重水平，生成ITS条形码的成本进一步降低。除了为四个有趣的地衣属的标本贡献新的ITS条形码外，本研究进一步强调了在馆藏标本上使用新测序技术为参考数据库生成DNA序列的潜力和挑战。
