title: Fungal diversity of Aeolus Cave soil after treatment with Trichoderma polysporum at concentration A, assessed via ITS2 sequencing, replicate 2
design: For microbial community analysis, DNA from 100 mg of untreated and Tp treated (105 and 106 conidia/g soil for AC and 105 conidia/g soil for BHM) soil samples (in duplicate) were extracted with the Powersoil DNA isolation kit (MO BIO Laboratories, Carlsbad, CA, USA). DNA concentrations were measured using a Nanodrop spectrophotometer ND 2000 (Nano-Drop Technologies, Wilmington, DE, USA). DNA libraries were prepared using a two-step PCR. In the first PCR, the primer set targeted the ITS2 region of the ribosomal RNA (rRNA) gene of fungi [22] and the hypervariable region V4 of the 16S rRNA gene of bacteria and archaea (http://www.earthmicrobiome.org/emp-standard-protocols/16s/). The same Illumina adaptors sequences were added to both fungal and bacterial/archaeal primers. Thus, the primer set used for 1st PCR for fungi had the following sequence: Forward primer 5 - TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAACTTTYRRCAAYGGATCWCT-3  (locus specific sequence in bold) and Reverse primer 5 - GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGCCTCCGCTTATTGATATGCTTAART -3  (locus specific sequence in bold). The primer set used for 1st PCR for bacteria/archaea had the following sequence: Forward primer 5 - TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA-3  (locus specific sequence in bold) and Reverse primer 5 - GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNVGGGTWTCTAAT-3  (locus specific sequence in bold). PCR reactions were carried out in a total volume of 25  l. Three microliters of extracted DNA (2.5 ng/ l) was added to the PCR reaction containing 13.3  l of sterile water, 0.2  l of bovine serum albumin (BSA), 2.5  l of Accutaq LA buffer, 2  l of dNTPs, 0.2  l of AccutaqTM LA DNA polymerase (Sigma-Aldrich, St. Louis, MO, USA) and 1  l each of fungal and bacterial/archaeal forward and reverse primers. All the PCR reactions were carried out in triplicate on a C1000 Touch Thermal Cycler (BioRad, Hercules, CA, USA). The thermocyling conditions for fungal specific PCR were initial denaturation at 95 C for 1 min, followed by 27 cycles of denaturation at 94 C for 30 s, annealing at 55 C for 1 min, extension at 68 C for 2 min, followed by final extension at 68 C for 5 min. For bacterial/archaeal specific PCR, thermocycling conditions were 95 C for 1 min, followed by 27 cycles of denaturation at 94 C for 30 s, annealing at 63 C for 1 min, extension at 68 C for 2 min, and a final extension at 68 C for 5 min. The first stage PCR products were run on the agarose gel to confirm the length of the respective bands. Triplicate samples were pooled to limit possible PCR artifacts. Agentcourt AMPure XP PCR purification kit (Beckman Coulter, Inc. Indianapolis, IN, USA) was used for the purification of the PCR products.The 2nd PCR step added dual indices (barcode) along with Illumina sequencing adaptors using the Nextera XT index kit (Illumina-16S Metagenomics protocol). PCR reactions were carried out in a total volume of 50  l, which contained 5  l of PCR product from 1st PCR and the 2x KAPA Hifi HotStart Ready mix. The reaction conditions were initial denaturation at 95 C for 3 min, followed by 8 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 30 s, and a final extension at 72 C for 5 min. All PCR reactions were performed in triplicate. The PCR products were cleaned using Agencourt AMPure XP PCR purification kit. DNA concentration was measured using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA) and the size was measured with an Agilent Tapestation using the D1000 High Sensitivity kit. The resulting DNA libraries were pooled, denatured and sequenced on MiSeq using MiSeq Reagent kit v3 (600 cycles) (Illumina Inc., San Diego, CA, USA) by the Wadsworth Center Advanced Sequencing Core Facility.