Strategies for sample labelling and library preparation in DNA metabarcoding studies.
Strategies for sample labelling and library preparation in DNA metabarcoding studies.
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DOI：
10.1111/1755-0998.13512
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发表时间：
2022-05
影响因子：
7.7
通讯作者：
Creer, Simon
中科院分区：
文献类型：
--
作者：
Bohmann, Kristine;Elbrecht, Vasco;Caroe, Christian;Bista, Iliana;Leese, Florian;Bunce, Michael;Yu, Douglas W.;Seymour, Mathew;Dumbrell, Alex J.;Creer, Simon
关键词：
从环境或大量标本中提取的DNA元条形码越来越多地用于基础和应用生物多样性研究中描述生物群，因为它的目标性质允许从许多样本中平行测序遗传标记。为了实现这一点，PCR扩增是用设计的引物进行的，目的是在分类群中针对分类信息标记，并在测序之前将样品特异性核苷酸标识符添加到扩增子中。后者可以将序列分配回它们起源的样本。核苷酸标识符可以在元条形码PCR和“文库制备”过程中添加，即在扩增子准备测序时添加。实现这种标签存在不同的策略。所有这些都有优点、挑战和局限性，其中一些可能导致误导性的结果，在最坏的情况下，会损害元条形码数据的保真度。考虑到使用元条形码解决的问题范围，确保数据生成稳健且适合所选目的对于寻求使用元条形码进行生物多样性评估的从业者至关重要。在这里，我们概述了在Illumina测序平台上进行元条形码研究中样品特异性标记和文库制备的三个主要工作流程；一步PCR，两步PCR和标记PCR。此外，我们提炼了研究人员寻求为其特定研究选择适当的元条形码策略的关键考虑因素。最终，通过深入了解不同元条形码工作流程的结果，我们希望进一步巩固元条形码作为评估生物多样性的工具的力量。
Metabarcoding of DNA extracted from environmental or bulk specimen samples is increasingly used to profile biota in basic and applied biodiversity research because of its targeted nature that allows sequencing of genetic markers from many samples in parallel. To achieve this, PCR amplification is carried out with primers designed to target a taxonomically informative marker within a taxonomic group, and sample‐specific nucleotide identifiers are added to the amplicons prior to sequencing. The latter enables assignment of the sequences back to the samples they originated from. Nucleotide identifiers can be added during the metabarcoding PCR and during “library preparation”, that is, when amplicons are prepared for sequencing. Different strategies to achieve this labelling exist. All have advantages, challenges and limitations, some of which can lead to misleading results, and in the worst case compromise the fidelity of the metabarcoding data. Given the range of questions addressed using metabarcoding, ensuring that data generation is robust and fit for the chosen purpose is critically important for practitioners seeking to employ metabarcoding for biodiversity assessments. Here, we present an overview of the three main workflows for sample‐specific labelling and library preparation in metabarcoding studies on Illumina sequencing platforms; one‐step PCR, two‐step PCR, and tagged PCR. Further, we distill the key considerations for researchers seeking to select an appropriate metabarcoding strategy for their specific study. Ultimately, by gaining insights into the consequences of different metabarcoding workflows, we hope to further consolidate the power of metabarcoding as a tool to assess biodiversity across a range of applications.
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影响因子：
16.6
作者：
Bista I;Carvalho GR;Walsh K;Seymour M;Hajibabaei M;Lallias D;Christmas M;Creer S
通讯作者：
Creer S
影响因子：
4.9
作者：
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通讯作者：
Alberdi, Antton
影响因子：
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作者：
Blackman, Rosetta C.;Maechler, Elvira;Deiner, Kristy
通讯作者：
Deiner, Kristy
影响因子：
4.9
作者：
Calvignac-Spencer, Sebastien;Merkel, Kevin;Leendertz, Fabian H.
通讯作者：
Leendertz, Fabian H.
影响因子：
7.7
作者：
Clarke, Laurence J.;Czechowski, Paul;Cooper, Alan
通讯作者：
Cooper, Alan