人工模拟条件下环境DNA宏条形码技术的定量分析初探

Preliminary study on the quantitative analysis of environmental DNA metabarcoding under ideal conditions

中文关键词: 环境DNA宏条形码  PCR引物偏好  高通量测序  线性函数关系  生物学调查

英文关键词: eDNA metabarcoding  PCR primer bias  High-throughput sequencing  Linear relationship  Biological investigation

中文摘要:

      近年来，环境DNA宏条形码技术(eDNA metabarcoding)在水生生态系统生物多样性评估等相关领域中得到广泛应用，因其具有快速测算群落中物种丰度的潜能，eDNA宏条形码技术成为资源保护和管理中颇具应用前景的调查工具。虽然大量证据表明eDNA高通量测序获得的reads数与自然环境中生物相对数量具有相关性，但一直不能得到明确的量化关系结果。eDNA的富集、扩增过程中的偏倚等诸多不确定因素，制约了该技术在生物资源调查领域的推广应用。假定水体中的eDNA全部回收，且PCR扩增时不存在引物偏倚性，这种理想状态下的水体中eDNA组成与其高通量测序reads数是否存在线性关系？为此，本研究在实验室可控条件下，选择2个同属近缘的凡纳滨对虾(Penaeus vannamei)和墨吉对虾(Penaeus merguiensis)，对其DNA样品进行不同比例混合，模拟从自然水体中富集到的eDNA复合样品，既保证了样品的回收率，又降低了引物偏倚的干扰。以此为模板，探究eDNA宏条形码技术检测种群相对数量的准确性。结果显示，当2个物种DNA模板浓度比例为1∶1时，高通量测序结果注释得到的2个物种reads数比值为13/24(墨吉对虾/凡纳滨对虾)，可见，即使是同属近缘种间依然存在轻微的引物偏倚现象，引物偏移率为1.5%。同时，根据7个实验组获得的高通量测序结果注释得到的2个物种reads数比值与对应模板中2个物种DNA浓度比值之间的线性回归分析表明，水体中eDNA组成与其高通量测序reads数间呈明显线性关系，即y=0.0716x+0.7043 (r²=0.9824)。综上所述，本研究为验证eDNA宏条形码技术监测水生生物资源量的可行性提供了直接证据，也为后续DNA宏条形码技术的定量研究提供了思路。

英文摘要:

      In recent years, environmental DNA (eDNA) metabarcoding has been widely used in the field of biological investigation, which includes species detection and biodiversity assessment. eDNA metabarcoding has the potential to rapidly assess species abundances in communities, making it a promising investigation tool in resource conservation and management. Although previous reports concerning eDNA metabarcoding found that the high-throughput sequencing (HTS) reads were related to the biomass in the natural environmental sample, a clear quantitative relationship was not found in this study. In the field and laboratory, the enrichment efficiency of eDNA is difficult to evaluate. Meanwhile, primer bias inevitably occurs in the eDNA amplification process, resulting in uncertainty in the eDNA HTS results, which restricts the application of eDNA metabarcoding for biological resource investigations. Assuming that the eDNA is completely recovered, and there is no primer bias during PCR amplification, an ideal state for deciphering whether there is a linear relationship between the eDNA and HTS reads is created. In this study, under controlled conditions in the laboratory, a sister group was selected (Penaeus vannamei and Penaeus merguiensis) and their DNA samples were mixed in different proportions to simulate eDNA samples enriched from natural waters. In this way, the recovery of the sample and primer bias was at optimal levels. Then, this eDNA template was used to explore the accuracy of eDNA metabarcoding in detecting species biomass. The results showed that when the concentration ratio of the DNA templates of two species was 1:1, the HTS ratio of the two species was 13/24 (P. merguiensis/ P. vannamei). Therefore, even between the closest relatives there is still a slight primer bias (primer migration rate: 1.5%). At the same time, the HTS results from the seven test groups showed an obvious linear relationship between the composition of eDNA in the water and the number of high-throughput sequencing reads, that is, y=0.0716x+0.7043 (r2=0.9824). In summary, this study provides direct evidence to verify the feasibility of eDNA metabarcoding in monitoring aquatic biological resources, and also provides ideas for the subsequent quantitative study of DNA metabarcoding.