An efficient early-pooling protocol for environmental DNA metabarcoding
An efficient early-pooling protocol for environmental DNA metabarcoding
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DOI：
10.1101/2022.02.15.480497
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发表时间：
2022-02
期刊：
影响因子：
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通讯作者：
Masayuki Ushio;Saori Furukawa;Hiroaki Murakami;R. Masuda;A. Nagano
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文献类型：
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作者：
Masayuki Ushio;Saori Furukawa;Hiroaki Murakami;R. Masuda;A. Nagano
环境DNA（Environmental DNA，eDNA）元条形码是一种将高通量测序和通用引物组合应用于eDNA分析的方法，已成为一种有效、全面的生物多样性监测方法。然而，执行eDNA元条形码仍然需要大量的金钱、劳动力和时间成本。在本研究中，我们评估了“早期汇集”方案（基于第一次PCR索引的方案）的性能，以降低eDNA元条形码文库制备的实验成本。具体地，我们进行了三个实验来测试第一PCR和第二PCR索引方案对通过eDNA元条形码显示的群落组成的影响，第一PCR后外切核酸酶纯化对索引跳跃的影响，以及PCR重复数和eDNA模板体积对检测到的OTU数的影响。通过分析来自3个自然水生生态系统的204个eDNA文库和1个模拟eDNA样品，我们表明：（i）第一次PCR索引不会导致eDNA元条形码结果的明显偏差，（ii）第一次PCR后的外切核酸酶纯化降低了索引跳跃的风险，和（iii）增加eDNA模板体积可以增加检测到的OTU的数量并减少检测到的群落组成的变化，也可以增加第一次PCR重复的数量。我们的研究结果表明，早期合并方案与后1st-PCR核酸外切酶纯化和DNA模板量的增加将降低索引跳跃的风险，耗材和试剂的成本，以及文库制备的处理时间，并且它产生与2nd-PCR索引方案相当的结果。因此，一旦确定了靶元条形码编码区并制备了一组索引的第一PCR引物，早期合并方案就提供了一种成本、劳动力和时间有效的方法来处理大量样品。
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 the present study, we assessed the performance of an “early-pooling” protocol (a protocol based on the 1st PCR indexing) to reduce the experimental costs of the library preparation for eDNA metabarcoding. Specifically, we performed three experiments to test the effects of 1st PCR and 2nd PCR indexing protocols on the community composition revealed by eDNA metabarcoding, of post-1st-PCR exonuclease purification on index-hopping, and of the number of PCR replicates and eDNA template volume on the number of detected OTUs. By analyzing 204 eDNA libraries from 3 natural aquatic ecosystems and 1 mock eDNA sample, we show that (i) the 1st PCR indexing does not cause clear biases in the outcomes of eDNA metabarcoding, (ii) post-1st-PCR exonuclease purification reduces the risk of index-hopping, and (iii) increasing the eDNA template volume can increase the number of detected OTUs and reduce the variations in detected community compositions, as can increasing the number of the 1st PCR replicates. Our results show that an early-pooling protocol with post-1st-PCR exonuclease purification and an increased amount of DNA template will reduce the risk of index-hopping, the costs for consumables and reagents, and the handling time in the library preparation, and that it produces comparable results to a 2nd-PCR-indexing protocol. Therefore, once a target metabarcoding region is determined and a set of indexed-1st-PCR primers is prepared, the early-pooling protocol provides a cost-, labor-, and time-efficient way to process a large number of samples.