An efficient early‐pooling protocol for environmental <scp>DNA</scp> metabarcoding
An efficient early‐pooling protocol for environmental <scp>DNA</scp> metabarcoding
复制标题
用于环境 <scp>DNA</scp> 元条形码的有效早期池协议
DOI：
10.1002/edn3.337
复制
发表时间：
2022
影响因子：
--
通讯作者：
Nagano Atsushi J.
中科院分区：
文献类型：
--
作者：
Ushio Masayuki;Furukawa Saori;Murakami Hiroaki;Masuda Reiji;Nagano Atsushi J.
环境DNA（eDNA）元条形码是一种将高通量测序和通用引物集应用于eDNA分析的方法，已成为高效、全面的生物多样性监测的一种有前途的方法。然而，执行eDNA元条形码仍然需要大量的金钱、人力和时间成本。在这项研究中，我们评估了“早期合并”方案（基于第一次PCR标记的方案）的性能，以降低eDNA元条形码文库制备的实验成本。具体而言，我们进行了三个实验来研究第一次PCR标记和第二次PCR索引方案对eDNA元条形码显示的群落组成的影响，第一次PCR后核酸外切酶纯化对标签跳跃的影响（对应于第二次PCR索引中的索引跳跃），以及PCR重复次数和eDNA模板体积对检测到的OTU数量的影响。对来自三个天然水生生态系统的204个eDNA文库和一个模拟eDNA样品的分析表明，（i）第一次PCR标记不会导致eDNA元条形码结果的明显偏差，（ii）第一次PCR后核酸外切酶纯化降低了标签跳跃的风险，以及（iii）增加eDNA模板体积可能会增加检测到的OTU的数量并减少检测到的群落组成的变化，类似于增加第一次PCR重复的数量。我们的研究结果表明，早期合并方案与第一次PCR核酸外切酶纯化和DNA模板量的增加降低了标签跳跃的风险，耗材和试剂的成本（除了许多标记的第一次PCR引物），以及文库制备的处理时间，并产生与第二次PCR索引方案相似的结果。因此，一旦选择了目标元条形码编码区并制备了一组带标签的第一PCR引物，早期合并方案就提供了一种用于处理大量样品的成本、劳动力和时间有效的方法。
Environmental DNA (eDNA) metabarcoding, a method that applies high‐throughput sequencing and universal primer sets to eDNA analysis, has been a promising approach for efficient, comprehensive biodiversity monitoring. However, significant money‐, labor‐, and time‐costs are still required for performing eDNA metabarcoding. In this study, we assessed the performance of an “early‐pooling” protocol (a protocol based on 1st PCR tagging) to reduce the experimental costs of library preparation for eDNA metabarcoding. Specifically, we performed three experiments to investigate the effects of 1st PCR‐tagging and 2nd PCR‐indexing protocols on the community composition revealed by eDNA metabarcoding, the effects of post‐1st PCR exonuclease purification on tag jumping (corresponds to index hopping in 2nd PCR indexing), and the effects of the number of PCR replicates and the eDNA template volume on the number of detected OTUs. Analyses of 204 eDNA libraries from three natural aquatic ecosystems and one mock eDNA sample showed that (i) 1st PCR tagging does not cause clear biases in the outcomes of eDNA metabarcoding, (ii) post‐1st PCR exonuclease purification reduces the risk of tag jumping, and (iii) increasing the eDNA template volume may increase the number of detected OTUs and reduce variations in the detected community compositions, similar to increasing the number of 1st PCR replicates. Our results show that an early‐pooling protocol with post‐1st PCR exonuclease purification and an increased amount of the DNA template reduces the risk of tag jumping, the costs for consumables and reagents (except for many tagged 1st PCR primers), and the handling time in library preparation, and produces similar results to a 2nd PCR‐indexing protocol. Therefore, once a target metabarcoding region is selected and a set of tagged‐1st PCR primers is prepared, the early‐pooling protocol provides a cost, labor, and time‐efficient approach for processing a large number of samples.