title: Amplicon sequencing (ITS) of hemlock root microbiome
design: An adapter sequence was added to each forward and reverse primer to make them compatible with Nextera XT indexes (Illumina). The initial polymerase chain reaction (PCR) consisted of 2 KAPA HiFi HotStart ReadyMix Taq (Roche, Indianapolis, Indiana, USA), 10 mol/L total for each forward primer combination, and 10 mol/L total for each reverse primer combination, with approximately 50 ng DNA. 16S rRNA and ITS2 PCRs were performed separately. Both reactions consisted of 3 min at 95C, followed by 25 cycles of 95C for 30 s, 55C for 30 s, and 72C for 30 s, with a final extension at 72C for 5 min. Successful PCR amplification was confirmed by running 4 L of PCR product on a 2% agarose gel. The PCR product was then purified by use of AMPure XP beads (Agencourt, Beverly, Massachusetts, USA). Nextera XT indexes were then ligated to the PCR products by use of a second, reduced cycle PCR, such that each sample had a unique combination of forward and reverse indexes. This reduced reaction consisted of three minutes at 95C, followed by eight cycles of 95C for 30 s, 55C for 30 s, and 72C for 30 s, with a final extension at 72C for five minutes. The products were purified again using AMPure XP beads. Samples were quantified on a NanoDrop spectrophotometer (Fisher Scientific) and pooled into an archaeal/bacterial pool and a fungal pool to approximately equal concentrations within each pool. Final product size and concentration were confirmed on a standard sensitivity Bioanalyzer (Agilent, Santa Clara, California, USA). Both bacterial and fungal samples were diluted to 4 mol/L, independently combined with 5% of a 4 mol/L PhiX adapterligated library control, and run pairedend on a v2, 500 cycle flow cell of an Illumina MiSeq sequencer.
library_name: 42-RS3-root-fungal