DNA_Soil_Core
. Briefly, bacterial (16S rRNA) and fungal (internal transcribed spacer, ITS) amplicon libraries were prepared using 8-bp molecular identification indices on both the forward and reverse primers. Polymerase chain reaction (PCR) of the V4 region of the 16S rRNA gene of the bacterial genomes was done using the modified 515F (5-GTGYCAGCMGCCGCGGTAA-3) [29] and 806R (5-GGACTACNVGGGTWTCTAAT-3) [30] primers and the following conditions: 98 C for 30 sec, 30 cycles of 98 C for 10 sec, 65 C for 10 sec, 72 C for 8 sec, and 72 C for 5 min. The ITS2 region of the fungal genomes was amplified using the fITS7 (5-GTGARTCATCGAATCTTTG-3) [31] and the ITS4 (5-TCCTCCGCTTATTGATATGC-3) [32] primers. Conditions for this amplification were as follows: 98 C for 30 sec, 30 cycles of 98 C for 10 sec, 60 C for 10 sec, 72 C for 8 sec, and 72 C for 5 min. Positive and negative controls were included in each round of PCR. Each of the triplicate PCRs consisted of 0.2 l of Phusion high-fidelity DNA polymerase, 4 l of 5X Phusion Green HF buffer, 0.4 l of deoxynucleotide triphosphates (10 M), 12.4 l of diethyl pyrocarbonate-water, 1 l each of forward and reverse primer (10 M), and 1 l of template DNA. PCRs were verified on 1.5% agarose gel, and successful reactions were combined in triplicate. Products were cleaned using Axygens AxyPrep Mag PCR Clean-up Kit according to manufacturer instructions (Axygen Biosciences, Union City, CA). Concentrations of the cleaned, amplified DNA were measured using a dsDNA HS assay kit on a Qubit 3.0 fluorometer (Invitrogen/ Life Technologies, Carlsbad, CA). Bacterial and fungal samples were combined at equimolar concentrations in separate libraries. Both libraries were sent to the University of Minnesotas Genomics Center (UMGC) for sequencing on the Illumina MiSeq platform with V2 chemistry (2 x 250 bp). Our final libraries did not include positive or negative controls for sequencing.