DNA metabarcoding has become a powerful approach for analysing complex communities from environmental samples, but there are still methodological challenges limiting its full potential. While conserved DNA markers, like 16S and 18S, often are not able to discriminate among closely related species, other more variable markers – like the fungal ITS region, may include considerable intraspecific variation, which can lead to oversplitting of species during DNA metabarcoding analyses. Here we assessed the effects of intraspecific sequence variation in DNA metabarcoding by analysing local populations of eleven fungal species. We investigated the allelic diversity of ITS2 haplotypes using both Sanger sequencing and high throughput sequencing (HTS) coupled with error correction with the software dada2. All the eleven species, except one, included some level of intraspecific variation in the ITS2 region. Overall, we observed a high correspondence between haplotypes generated by Sanger sequencing and HTS, with the exception of a few additional haplotypes detected using either approach. These extra haplotypes, typically occurring in low frequencies, were probably due to PCR and sequencing errors or intragenomic variation in the rDNA region. The presence of intraspecific (and possibly intragenomic) variation in ITS2 suggest that haplotypes (or ASVs) should not be used as basic units in ITS‐based fungal community analyses, but an extra clustering step is needed to approach species‐level resolution.

中文翻译：

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DNA元条形码过程中种内序列变异的影响：11种真菌的案例研究

DNA元条形码已成为从环境样本中分析复杂群落的强大方法，但仍然存在限制其全部潜力的方法学挑战。虽然保守的 DNA 标记，如 16S 和 18S，通常无法区分密切相关的物种，但其他更可变的标记——如真菌 ITS 区域，可能包括相当大的种内变异，这可能导致 DNA 元条形码分析期间物种过度分裂。在这里，我们通过分析 11 种真菌物种的本地种群来评估种内序列变异对 DNA 宏条形码的影响。我们使用 Sanger 测序和高通量测序 (HTS) 以及使用软件dada2 进行纠错，研究了 ITS2 单倍型的等位基因多样性. 除一种外，所有 11 种物种都在 ITS2 区域包含一定水平的种内变异。总体而言，我们观察到 Sanger 测序和 HTS 生成的单倍型之间的高度对应，但使用任一方法检测到的一些额外单倍型除外。这些额外的单倍型通常以低频率出现，可能是由于 PCR 和测序错误或 rDNA 区域的基因组内变异。ITS2 中存在种内（也可能是基因组内）变异表明单倍型（或 ASV）不应用作基于 ITS 的真菌群落分析的基本单位，但需要额外的聚类步骤来接近物种级别的分辨率。