title: Metabarcoding soil fungi
design: Soil samples were processed by RTL Genomics, Lubbock, Texas, USA (http://rtlgenomics.com/) using RTL Genomics' Microbial Diversity Assay with a target of an average of at least 10,000 reads per sample. Extractions were performed using a Qiagen MagAttract PowerSoil DNA KF Kit. For soil samples, each whole sample was agitated and a pea-sized amount was added to the Qiagen MagAttract PowerSoil \acr{DNA} \acr{KF} Kit bead plate. Samples were amplified for sequencing in a two-step process using the ITS3F (GCATCGATGAAGAACGCAGC) and ITS4R TCCTCCGCTTATTGATATGC} primer pair, amplifying part of the ITS2 region. The forward primer was constructed with (5'-3') the Illumina i5 sequencing primer (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG) and the ITS3F primer. The reverse primer was constructed with (5'-3') the Illumina i7 sequencing primer (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG) and the ITS4R primer. Products from the first stage amplification were added to a second PCR based on qualitatively determined concentrations. Primers for the second PCR were designed based on the Illumina Nextera PCR primers as follows: Forward - AATGATACGGCGACCACCGAGATCTACAC[i5index]TCGTCGGCAGCGTC and Reverse - CAAGCAGAAGACGGCATACGAGAT[i7index]GTCTCGTGGGCTCGG. The second stage amplification was run the same as the first stage except for 10 cycles. Amplification products were visualized with eGels (Life Technologies, Grand Island, New York). Products were then pooled equimolar and each pool was size selected in two rounds using Agencourt AMPure XP (BeckmanCoulter, Indianapolis, Indiana) in a 0.75 ratio for both rounds. Size selected pools were then quantified using the Quibit 2.0 fluorometer (Life Technologies) and loaded on an Illumina MiSeq (Illumina, Inc. San Diego, California) 2x300 flow cell at 10 pM.
library_name: 2017MLB100-1