Scrutinizing key steps for reliable metabarcoding of environmental samples

1. Metabarcoding of environmental samples has many challenges and limitations that require carefully considered laboratory and analysis pipelines to ensure reliable results. We explore how decisions regarding study design, laboratory work and bioinformatic processing affect the final results, and provide guidelines for reliable study of environmental samples. 2. We evaluate the performance of four primer sets targeting COI and 16S regions characterising arthropod diversity in bat faecal samples, and investigate how metabarcoding results are affected by parameters including: i) number of PCR replicates per sample, ii) sequencing depth, iii) PCR replicate processing strategy (i.e. either additively, by combining the sequences obtained from the PCR replicates, or restrictively, by only retaining sequences that occur in multiple PCR replicates for each sample), iv) minimum copy number for sequences to be retained, v) chimera removal, and vi) similarity thresholds for OTU clustering. Lastly,...

1. 环境样本的元条形码（metabarcoding）技术面临诸多挑战与局限，需经审慎规划的实验室操作与分析流程，方可获得可靠结果。本研究探讨了研究设计、实验室工作流程及生物信息学处理环节的各类决策对最终结果的影响，并为环境样本的可靠研究提供指导方案。
2. 本研究针对蝙蝠粪便样本中节肢动物多样性特征的COI与16S区域，评估了4对引物的扩增效果，并探究了以下参数对元条形码分析结果的影响：① 单样本聚合酶链式反应（Polymerase Chain Reaction, PCR）重复次数；② 测序深度；③ PCR重复产物的处理策略（即加法策略：合并所有PCR重复得到的序列；或减法策略：仅保留在单个样本的多次PCR重复中均出现的序列）；④ 序列保留的最小拷贝数阈值；⑤ 嵌合体移除；⑥ 操作分类单元（Operational Taxonomic Unit, OTU）聚类的相似性阈值。最后，……