FROM SAMPLING TO TAXONOMY - Critical Control Points in DNA Metabarcoding Workflows

Amplicon-based metagenomics is an effective tool for the study of various biological communities - from bacteria to higher eukaryotes – in various environments such as the animal/human body, soil, water, and other ecosystems. However, achieving reliable and reproducible results is methodologically demanding.

As a provider of sequencing services, we gain - through cooperation with customers from a wide spectrum of fields - a unique overview of the methodological pitfalls and critical control points that can significantly influence the quality and reliability of research project outputs. Our goal is the systematic optimization of at least a part of the many factors which, in sum across the entire metagenomics workflow, significantly complicate the interpretation of analysis results.

Using specific examples, we demonstrate that any change of the workflow such as sample collection and preservation, DNA extraction based on the starting material (stool, soil, water, etc.) and project objectives (G+/G- bacteria, fungal communities, or higher eukaryotes), and other following wetlab factors, e.g. PCR specifications - target regions, primers and their modifications, specific reagents (e.g., polymerase) and their concentrations, or thermal profiles, can significantly affect the result (the relative abundance of the specific organisms within the sample).

We demonstrate that also sequencing and data analysis have great impact on the output. The key factors are NGS sequencing platform selection (short versus long reads), bioinformatic processing strategy, pipeline and software implementation and reference database selection.

Standardizing the determination of various community types via amplicon sequencing represents a complex task that requires careful and systematic methodology optimization. A key element for verifying the correctness of the procedure is the use of clearly defined mock community standards. Furthermore, to ensure the comparability of results between individual facilities, it is essential to engage in inter-laboratory quality controls or to participate in official external quality assessment (EQA) programs guaranteed by a recognized authority.