title: MiSeq of eDNA 16S gene for macroinvertebrate diversity
design: Three metabarcoding markers targeting the mtDNA 16S gene, designated as: (1) Copepod  Clarke et al., 2017, (2) Insect  Epp et al., 2012, and (3) Mollusk  Klymus et al., 2017, were used to analyze the macroinvertebrate communities of the 15 sites. Each primer was attached to a spacer region, which increased library nucleotide (nt) diversity of sequence reads and enhanced cluster formation, improving sequencing quality (Klymus et al., 2017; Marshall & Stepien, 2019). All markers followed the same library preparation: the 1st PCR protocol included 1X PCR buffer, 0.3mM dNTPs, 0.5M of each primer, an additional 1.5mM MgCl2, 5U AllTaq (Qiagen), 5l template DNA, and ddH20 to total 50l. Conditions were: 2min initial denaturation at 95C, followed by 40 cycles of 95C for 5sec, TA for 15sec, and 72C for 10sec, with no final extension. All first step PCR products were processed with a 0.7X HighPrepTM bead clean-up (MagBio Genomics, kit AC-60050), yielding the template for the subsequent step. The 2nd and final PCR incorporated Nextera paired-end indices (Illumina, kit FC-121-1011) p5/p7 adaptor sequences to allow the sample to bind onto the Illumina MiSeq flow cell and incorporated eight bases that allowed samples to be identified in the raw read data. The final 25l PCR reaction contained a variable volume of ddH20, 1X PCR buffer, 0.2mM dNTPs, 2.5l of each primer, 1.57U NEB Hotstart Taq polymerase (New England Biolabs Inc.), and 2.5L from the previous PCR cleanup. Conditions were: 30sec initial denaturation at 95C, followed by 8 cycles at 95C for 30sec, 55C for 30sec, and 68C for 1min, with a final 2min 68C extension. PCR products were sized and quantified on a 2100 Bioanalyzer (Agilent Technologies) prior to Illumina MiSeq analysis conducted at the Ohio State Universitys Molecular and Cellular Imaging Center in Wooster, OH (http://mcic.osu.edu/). Pooled product concentrations were measured on a Qubit fluorometer (Invitrogen), and the pooled samples were run with 2X 300nt V3 Illumina MiSeq chemistry. An additional 4050% PhiX DNA spike-in control was added to improve data quality of low nt diversity samples.