Data from: Diversity measures in environmental sequences are highly dependent on alignment quality—data from ITS and new LSU primers targeting basidiomycetes

The ribosomal DNA comprised of the ITS1-5.8S-ITS2 regions is widely used as a fungal marker in molecular ecology and systematics but cannot be aligned with confidence across genetically distant taxa. In order to study the diversity of Agaricomycotina in forest soils, we designed primers targeting the more alignable 28S (LSU) gene, which should be more useful for phylogenetic analyses of the detected taxa. This paper compares the performance of the established ITS1F/4B primer pair, which targets basidiomycetes, to that of two new pairs. Key factors in the comparison were the diversity covered, off-target amplification, rarefaction at different Operational Taxonomic Unit (OTU) cutoff levels, sensitivity of the method used to process the alignment to missing data and insecure positional homology, and the congruence of monophyletic clades with OTU assignments and BLAST-derived OTU names. The ITS primer pair yielded no off-target amplification but also exhibited the least fidelity to the expected phylogenetic groups. The LSU primers give complementary pictures of diversity, but were more sensitive to modifications of the alignment such as the removal of difficult-to align stretches. The LSU primers also yielded greater numbers of singletons but also had a greater tendency to produce OTUs containing sequences from a wider variety of species as judged by BLAST similarity. We introduced some new parameters to describe alignment heterogeneity based on Shannon entropy and the extent and contents of the OTUs in a phylogenetic tree space. Our results suggest that ITS should not be used when calculating phylogenetic trees from genetically distant sequences obtained from environmental DNA extractions and that it is inadvisable to define OTUs on the basis of very heterogeneous alignments.由内部转录间隔区1-5.8S-内部转录间隔区2（ITS1-5.8S-ITS2）区域组成的核糖体DNA（ribosomal DNA）在分子生态学和系统分类学中被广泛用作真菌标记基因，但在遗传距离较远的类群间难以进行可靠的序列比对。为研究森林土壤中伞菌亚门（Agaricomycotina）的多样性，我们设计了靶向比对性更强的28S（LSU）基因的引物，该基因更适用于对检测到的类群进行系统发育分析。本研究对比了已有的靶向担子菌门（basidiomycetes）的ITS1F/4B引物对与两款新型引物对的性能。对比的核心指标包括：覆盖的物种多样性、非靶标扩增情况、不同操作分类单元（Operational Taxonomic Unit, OTU）阈值水平下的稀疏化分析、比对处理方法对缺失数据和不可靠位置同源性的敏感性，以及单系支与OTU分配、BLAST衍生OTU名称的一致性。ITS引物对未产生非靶标扩增，但对预期系统发育类群的保真度最低。LSU引物对可提供互补的多样性观测结果，但对比对修饰（如移除难以比对的区段）更为敏感。LSU引物对还产生了更多的单例序列（singletons），且根据BLAST相似性判断，其生成的OTU更易包含来自更多不同物种的序列。我们引入了基于香农熵（Shannon entropy）的比对异质性新参数，以及系统发育树空间中OTU的范围与组成。研究结果表明，当从环境DNA提取物获得的遗传距离较远的序列构建系统发育树时，不应使用ITS区域；且基于异质性极强的比对结果定义OTU并不可取。