Center for Marine Environmental Studies, Ehime University, Matsuyama, Ehime, Japan.
Faculty of Engineering, Graduate School of Science and Engineering, Ehime University, Matsuyama, Ehime, Japan.
PLoS One. 2023 Jul 24;18(7):e0289056. doi: 10.1371/journal.pone.0289056. eCollection 2023.
Metabarcoding is a molecular-based tool capable of large quantity high-throughput species identification from bulk samples that is a faster and more cost-effective alternative to conventional DNA-sequencing approaches. Still, further exploration and assessment of the laboratory and bioinformatics strategies are required to unlock the potential of metabarcoding-based inference of haplotype information. In this study, we assessed the inference of freshwater macroinvertebrate haplotypes from metabarcoding data in a mock sample. We also examined the influence of DNA template concentration and PCR cycle on detecting true and spurious haplotypes. We tested this strategy on a mock sample containing twenty individuals from four species with known haplotypes based on the 658-bp Folmer region of the mitochondrial cytochrome c oxidase gene. We recovered fourteen zero-radius operational taxonomic units (zOTUs) of 421-bp length, with twelve zOTUs having a 100% match with the Sanger haplotype sequences. High-quality reads relatively increased with increasing PCR cycles, and the relative abundance of each zOTU was consistent for each cycle. This suggests that increasing the PCR cycles from 24 to 64 did not affect the relative abundance of each zOTU. As metabarcoding becomes more established and laboratory protocols and bioinformatic pipelines are continuously being developed, our study demonstrated the method's ability to infer intraspecific variability while highlighting the challenges that must be addressed before its eventual application for population genetic studies.
代谢条形码是一种基于分子的工具，能够从大量样本中快速、高效地识别物种，是传统 DNA 测序方法的一种更快、更经济有效的替代方法。然而，为了充分发挥基于代谢条形码推断单倍型信息的潜力，还需要进一步探索和评估实验室和生物信息学策略。在这项研究中，我们评估了从模拟样本中的代谢条形码数据推断淡水大型无脊椎动物单倍型的能力。我们还研究了 DNA 模板浓度和 PCR 循环对检测真实和虚假单倍型的影响。我们在一个包含四个已知单倍型物种的 20 个个体的模拟样本中测试了这种策略，该模拟样本基于线粒体细胞色素 c 氧化酶基因的 658-bp Folmer 区。我们恢复了 14 个长度为 421-bp 的零半径操作分类单位（zOTU），其中 12 个 zOTU 与 Sanger 单倍型序列完全匹配。高质量的读数随着 PCR 循环的增加而相对增加，并且每个循环的每个 zOTU 的相对丰度是一致的。这表明，将 PCR 循环从 24 增加到 64 不会影响每个 zOTU 的相对丰度。随着代谢条形码技术的不断发展和实验室协议以及生物信息学流程的不断完善，我们的研究证明了该方法推断种内变异性的能力，同时突出了在最终将其应用于种群遗传学研究之前必须解决的挑战。
