Diversity of Wadden Sea macrofauna and meiofauna communities highest in DNA from extractions preceded by cell lysis

Abstract

Metabarcoding of genetic material in environmental samples has increasingly been employed as a means to assess biodiversity, also of marine benthic communities. Current protocols employed to extract DNA from benthic samples and subsequent bioinformatics pipelines differ considerably. The present study compares three commonly deployed metabarcoding approaches against a morphological approach to assess benthic biodiversity in an intertidal bay in the Dutch Wadden Sea. Environmental DNA was extracted using three different approaches; extraction of extracellular DNA, extraction preceded by cell lysis of a sieved fraction of the sediment, and extraction of DNA directly from small amounts of sediment. DNA extractions after lysis of sieved sediment fractions best recovered the macrofauna diversity whereas direct DNA extraction of small amounts of sediment best recovered the meiofauna diversity. Extractions of extracellular DNA yielded the lowest number of OTUs per sample and hence an incomplete view of benthic biodiversity. An assessment of different bioinformatic pipelines and parameters was conducted using a mock sample with a known species composition. The RDP classifier performed better than BLAST for taxonomic assignment of the samples in this study. Novel metabarcodes obtained from local specimens were added to the SILVA 18S rRNA database to improve taxonomic assignment. This study provides recommendations for a general metabarcoding protocol for marine benthic surveys in the Wadden Sea.

A Molecular Approach to Explore the Background Benthic Fauna Around a Hydrothermal Vent and Their Larvae: Implications for Future Mining of Deep-Sea SMS Deposits

“…However, the detection of macrofauna species might be biased as the release and fate of eDNA differs per species (Barnes and Turner, 2016). In this study, DNA was isolated from a small amount of unsieved sediment, to include all smaller specimen and an additional DNA extraction on the sieved sediment fraction was used, to improve the detection of macrofauna species (Brannock and Halanych, 2015;Klunder et al, 2019). Therefore, the sieve residues from the sieved samples were cryodesiccated and ground in an agate mortar using liquid nitrogen.…”

Cascabel: A Scalable and Versatile Amplicon Sequence Data Analysis Pipeline Delivering Reproducible and Documented Results

“…Cascabel has been developed at the Royal Netherlands Institute for Sea Research (NIOZ) to facilitate, unify and easily track data provenance of amplicon sequence data analyses. Apparent advantages of using this pipeline compared to custom scripts are that the individual steps of the pipeline have been tested by many members of the community at the NIOZ who are experienced in amplicon sequencing data analyses (van Bleijswijk et al, 2015;Balzano et al, 2018;Besseling et al, 2019;Klunder et al, 2019), and therefore should contain fewer mistakes than scripts that were written for a specific analysis by one person. Moreover, community knowledge and experience have created a workflow which is probably more comprehensive and powerful than one that was created by a single person.…”

Cascabel: a flexible, scalable and easy-to-use amplicon sequence data analysis pipeline

“…Cascabel has been developed at the Royal Netherlands Institute for Sea Research (NIOZ) to facilitate, unify and easily track data provenance of amplicon sequence data analyses. Apparent advantages of using this pipeline compared to custom scripts are that the individual steps of the pipeline have been tested by many members of the community at the NIOZ who are experienced in amplicon sequencing data analyses (34)(35)(36)(37), and therefore should contain fewer mistakes than scripts that were written for a specific analysis by one person. Moreover, community knowledge and experience have created a workflow which is probably more comprehensive and powerful than one that was created by a single person.…”