title: eDNA nodule metabarcoding of marine eukaryotes in the NORI-D license area of the CCZ
design: Nodules were extracted using the FastDNA 50mL SPIN kit for Soil (MPBio, CA, United States) following the manufacturers protocol with modifications for elongated vortexing and incubation times. The total mass of nodules for a given core was weighed, and a single nodule was isolated and weighed independently. A total of two nodule extractions were performed for each core, one of an isolated nodule weighing at least 10g, and the conglomerated remaining nodules. Nodule samples were defrosted, ground with a ceramic pestle, and thoroughly mixed with a sterile spatula prior to subsampling.Eukaryotic communities were characterized by amplicon sequencing using a fragment (ca. 350 bp) of the mitochondrial COI gene. For COI, the universal metazoan primers mlCOIintF: 5-GGW ACW GGW TGA ACW GTW TAY CCY CC-3 and jgHCO2198: 5-TAI ACY TCI GGR TGI CCR AAR AAY CA-3 (Leray et al., 2013). Primers were modified to include IlluminaTM overhang adaptors. PCR reactions consisted of 12.5 L of MyFi Mix (Bioline), 1 L of each primer (10 M), 1 L of bovine serum albumin (300 M; BSA; Sigma), 1-3 L of template DNA (min 2.5 ng/L per reaction) with ddH2O added to reach a total volume of 25 L. Cycling conditions for COI were: 95C for 3 min, followed by 45 cycles of 95C for 15 s, 50C for 30 s, 72C for 15 s, with a final extension step at 72C for 1 min. Each PCR experiment included a negative control (no template DNA) to ensure there was no contamination of reagents. Amplicons were purified with AMPure XP beads (Beckman Coulter, Indiana, USA) and quantified using a Qubit Fluorometer (Life Technologies) following the manufacturers instructions. DNA concentration was normalised to 2 ng/L with ddH2O. Samples, including two negative controls, were given to the Advanced Studies in Genomics Proteomics and Bioinformatics (ASGPB) of the University of Hawaii at Manoa (HI, USA) for indexing with the NexteraTM DNA library Prep Kit (Illumina, California, USA). Samples were pooled into two libraries and sequenced on two MiSeq IlluminaTM lanes using V3 chemistry and paired end sequencing (2300 bp). Each library contained a negative control to assess potential contamination during the second PCR step and sequencing.