title: Indian Creek 2A
design: We extracted DNA from water filters using the PowerWater DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA) following the manufacturers instructions. We amplified the bacterial 18S rRNA region using primer pair (F- AGCTTGTCTCAAAGATTAAGC, R  TCCCTCTCCGGAATCGAACCC) with the attachment of connector sequences (Hernndez-Gmez et al., 2017). We ran each sample in triplicate, and each reaction consisted of 5 L of template DNA, 7.5 L of MyTaq Master Mix (Bioline, Tauton, MA), 1 L of 10 mM forward and reverse primers, and 1.5 L of PCR water (MoBio Laboratories Inc., Carlsbad, CA) for a total of 15 L per reaction. PCR conditions consisted of 95C for 2 minutes, 30 cycles of 94C for 45 seconds, 52C for 60 seconds, and 72C for 90 seconds, followed by 72C for 10 minutes. We pooled amplicon triplicates and cleaned the products using the UltraClean PCR Clean-up kit (MoBio Laboratories Inc., Carlsbad, CA). We performed a second PCR to add-on dual-index barcodes connected to Illumina sequencing adaptors (Hernndez-Gmez et al., 2017) to the ends of amplicons. The PCR consisted of 3 L of clean amplicons, 5 L of MyTaq Master Mix, 1 L of forward and reverse barcode primers, and 1 L of water for a total of 10 L reactions. PCR conditions consisted of 95C for 2 minutes, 7 cycles of 94C for 45 seconds, 65C for 60 seconds, and 72C for 90 seconds, followed by 72C for 10 minutes. We quantified the PCR products using a Qubit Fluorometer (Invitrogen Corp, Carlsbad, CA), pooled samples in equimolar amounts, and cleaned the sample pool using the UltraClean PCR Clean-Up kit. The sample pool was sequenced on a MiSeq machine (Illumina Inc. San Diego, CA) using the Reagent Kit V2 to produce 250 bp paired end reads.