A flexible pipeline combining clustering and correction tools for prokaryotic and eukaryotic metabarcoding

Abstract

SUPPLEMENTAL FIGURESFigure S 1. Rarefaction curves in 14 deep-sea sediment sites and 2 mock control samples for metabarcoding results of the COI (left) and 18S (middle), and 16S (right) marker genes for the ASV (top) and an OTU (bottom) datasets, showing a plateau is reached in all samples.

An Assessment of Environmental Metabarcoding Protocols Aiming at Favoring Contemporary Biodiversity in Inventories of Deep-Sea Communities

“…Four primer pairs were used to amplify one mitochondrial and three rRNA barcode loci targeting metazoans (COI, 18S-V1), micro-eukaryotes (18S-V4), and prokaryotes (16S-V4V5 for homogeneity; Supplementary Table S3). Two metazoan mock communities (detailed in Brandt et al, 2020) were included for 18S-V1 and COI. For each sample and marker, triplicate amplicon libraries (see Supporting Information for amplification details) were prepared by ligation of Illumina adapters on 100 ng of amplicons following the Kapa Hifi HotStart NGS Library Amplification Kit (Kapa Biosystems, Wilmington, MA, United States).…”

“…All bioinformatic analyses were performed using a Unix shell script (Brandt et al, 2020), available on Gitlab 1 , on a home-based cluster (DATARMOR, Ifremer). The details of the pipeline, along with specific parameters used for all metabarcoding markers, are given in Supplementary Table S4 and in Brandt et al (2020).…”

“…All bioinformatic analyses were performed using a Unix shell script (Brandt et al, 2020), available on Gitlab 1 , on a home-based cluster (DATARMOR, Ifremer). The details of the pipeline, along with specific parameters used for all metabarcoding markers, are given in Supplementary Table S4 and in Brandt et al (2020). Pairs of Illumina reads were corrected with DADA2 v.1.10 (Callahan et al, 2016) following the online tutorial for paired-end data 2 and delivered inventories of amplicon sequence variants (ASVs).…”

Exploring Environmental DNA (eDNA) to Assess Biodiversity of Hard Substratum Faunal Communities on the Lucky Strike Vent Field (Mid-Atlantic Ridge) and Investigate Recolonization Dynamics After an Induced Disturbance

“…In attempts to improve the efficiency of molecular methods for detecting living communities, as well as provide more accurate assessments of biodiversity over time, studies have used treatments during the DNA extraction phase to separate intra and extracellular DNA within environmental samples (Wagner et al, 2008;Taberlet et al, 2012b;Seidel et al, 2017). Additional strategies have extracted eRNA of microorganisms, as eRNA is less stable and degrades more rapidly than eDNA and can thus improve biodiversity assessment of contemporary communities (Barnes and Turner, 2016;Pochon et al, 2017;Laroche et al, 2018;Brandt et al, 2019). Next, the incompleteness of public databases used for taxonomic assignment during high-throughput sequencing projects, as well as the selection of genetic markers, heavily influence eDNA studies.…”

“…To address these shortcomings, the best strategy includes the continued inclusion of well curated data from hydrothermal vent and deep-sea fauna into public inventories, to which we have contributed in the present study via our barcoding efforts. Furthermore, the use of a standardized bioinformatic pipeline targeting poorly surveyed marine habitats that outlines specific parameters and filters would allow more precise and uniform results and this effort is currently underway (Brandt et al, 2019). These outcomes also emphasize the importance of collaborative efforts of morphological and molecular approaches, at least during the initial stages of community characterization (Cowart et al, 2015).…”