Metabarcoding of a mock community of soil invertebrates: DNA extraction, false-positives, and data filtration

Metabarcoding is a powerful tool widely used to analyze soil communities but methodological difficulties can introduce biases, resulting in inaccurate estimation of species diversity. We evaluated five DNA extraction methods, two PCR programs, and three post-bioinformatic filtering techniques (multiple databases, filtering based on the level of similarity and on the number of reads obtained) using a mock soil community of invertebrates containing 24 species, 23 genera, and 15 families of arthropods and annelids. The choice of DNA extraction kit significantly influenced the accuracy of the metabarcoding results. Kits developed for the isolation of DNA from soil and alkaline lysis without purification showed worse results compared to those designed for isolating DNA from animal tissues. Increasing the annealing temperature of primers resulted in an increased number of false positives. On the contrary, the use of post-bioinformatic filtering dramatically increased the proportion of true positives, reaching 100% correctness for families, more than 90 % for genera and more than 75% for species forming the mock community. Thus, an acceptable level of accuracy may be achieved in field surveys with relatively little time and effort.

元条形码（Metabarcoding）是广泛应用于土壤群落分析的有力工具，但方法学层面的难点易引入分析偏倚，进而导致物种多样性估算结果失准。本研究以包含24个物种、23个属、15个科的节肢动物与环节动物的无脊椎动物模拟土壤群落为研究对象，评估了5种DNA提取方法、2套聚合酶链式反应（PCR）程序以及3种生物信息学后过滤技术（涵盖多数据库比对、基于序列相似性的过滤、基于获得读段（reads）数量的过滤）。研究表明，DNA提取试剂盒的选择对元条形码分析结果的准确性存在显著影响：针对土壤样本DNA提取开发的试剂盒，以及未设置纯化步骤的碱裂解法试剂盒，其分析效果均逊于针对动物组织DNA提取设计的试剂盒。提升引物退火温度会导致假阳性序列数量增加；与之相反，应用生物信息学后过滤技术可显著提升真阳性序列占比，针对本次模拟群落的分析准确率可达科分类水平100%、属分类水平90%以上、物种分类水平75%以上。因此，仅需投入相对有限的时间与人力，即可在野外调查中达到可接受的分析准确率水平。