title: eDNA nodule metabarcoding extraction blank
design: Extraction blank using FastDNA 50mL SPIN kit for Soil (MPBio, CA, United States) following the manufacturers protocol with modifications for elongated vortexing and incubation times. Eukaryotic communities were characterized by amplicon sequencing using the V4 region of the 18S rRNA gene (approximately 450 base pairs [bp]). For 18S rRNA, the eukaryotic forward Uni18SF: 5-AGG GCA AKY CTG GTG CCA GC-3 and reverse primers Uni18SR: 5-GRC GGT ATC TRA TCG YCT T-3 primers (Zhan et al., 2013) were used. Primers were modified to include IlluminaTM overhang adaptors. The 18S V4 PCR reactions consisted of 12.5 L of MyFi Mix (Bioline), 0.5 L of each primer (10 M), 0.6 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. The reaction cycling conditions were: 95C for 3 min, followed by 35 cycles of 95C for 15 s, 55C for 30 s, 72C for 15 s, with a final extension step at 72C for 1 min. PCR was run in duplicate and 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.