National Institute of Oceanography, Department of Biology and Biotechnology, Israel Oceanographic & Limnological Research, P.O.B 9753, Haifa 3109701, Israel; Leon H. Charney School of Marine Sciences, Department of Marine Biology, University of Haifa, 199 Aba Koushy Ave., Mount Carmel, Haifa 3498838, Israel.
National Institute of Oceanography, Department of Biology and Biotechnology, Israel Oceanographic & Limnological Research, P.O.B 9753, Haifa 3109701, Israel; Leon H. Charney School of Marine Sciences, Department of Marine Biology, University of Haifa, 199 Aba Koushy Ave., Mount Carmel, Haifa 3498838, Israel.
Mar Genomics. 2022 Oct;65:100980. doi: 10.1016/j.margen.2022.100980. Epub 2022 Aug 10.
The present study is aimed at implementing the morphological identification-free amplicon sequence variant (ASV) concept for describing meiofaunal species composition, while strongly indicating reasonable compatibility with the underlying species. A primer pair was constructed and demonstrated to PCR amplify a 470-490 bp 18S barcode from a variety of meiofaunal taxa, high throughput sequenced using the Illumina 300 × 2 bps platform. Sixteen 18S multi-species HTS assemblies were created from meiofaunal samples and merged to one assembly of ~2,150,000 reads. Five quality scores (q = 35, 30, 25, 20, 15) were implemented to filter five 18S barcode assemblies, which served as inputs for the DADA2 software, ending with five reference ASV libraries. Each of these libraries was clustered, applying 3% dissimilarity threshold, revealed an average number of 1.38 ± 0.078 ASVs / cluster. Hence, demonstrating high level of ASV uniqueness. The libraries which were based on q ≤ 25 reached a near-asymptote number of ASVs which together with the low average number of ASVs / cluster, strongly indicated fair representation of the actual number of the underlying species. Hence, the q = 25 library was selected to be used as metabarcoding reference library. It contained 461 ASVs and 342-3% clusters with average number of 1.34 ± 1.036 ASV / cluster and their BLASTN annotation elucidated a variety of expected meiofaunal taxa. The sixteen assemblies of sample-specific paired reads were mapped to this reference library and sample ASV profiles, namely the list of ASVs and their proportional copy numbers were created and clustered.
本研究旨在实施形态识别自由扩增子序列变异 (ASV) 概念来描述后生动物的物种组成，同时强烈表明与潜在物种具有合理的兼容性。构建了一对引物，并证明其能够从各种后生动物类群中扩增出 470-490 bp 的 18S 条形码，然后使用 Illumina 300×2bps 平台进行高通量测序。从后生动物样本中创建了 16 个 18S 多物种 HTS 组装，并合并为一个约 2,150,000 个读取的组装。实施了五个质量分数 (q=35、30、25、20、15) 来过滤五个 18S 条形码组装，这些组装作为 DADA2 软件的输入，最终得到五个参考 ASV 文库。每个文库都进行聚类，应用 3%的不相似性阈值，揭示平均每个聚类有 1.38±0.078 个 ASV。因此，表现出高度的 ASV 独特性。基于 q≤25 的文库达到了接近 ASV 数量的渐近线，再加上每个聚类的平均 ASV 数量较少，强烈表明了潜在物种的实际数量有很好的代表性。因此，选择 q=25 的文库作为 metabarcoding 参考文库。它包含 461 个 ASV 和 342-3%的聚类，平均每个聚类有 1.34±1.036 个 ASV，其 BLASTN 注释阐明了各种预期的后生动物类群。十六个样本特定的配对读取组装被映射到这个参考文库上，并创建了样本 ASV 图谱，即 ASV 列表及其比例拷贝数，并进行了聚类。
