Potential for passive mitigation of coal-derived acid mine drainage in abandoned stream channels
DNA was sequenced using 16S rRNA gene amplicon sequencing that was conducted on MiSeq Illumina using bacterial tag-encoded (bTEDFAQ) by Molecular Research Laboratories. HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA) was used for 28-step single cycle PCR with following parameters: initial denaturation at 94 C for 3 min, 28 cycles of 94 C for 30 s, 53 C for 40 s, and 72 C for 1 min, and a final elongation step at 72 C for 5 min. Amplified PCR products were further mixed in equal concentration and purified with calibrated Agencourt AMPure XP beads. Amplicon sequencing was done at MiSeq Illumina by Molecular Research Laboratories, LP (Shallowater, TX). After removal of barcodes and primers from Illumina derived sequences, all sequences that were less than 150 bp, included chimeras, ambiguous base calls, and homopolymers >6 bp were discarded.
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