title: Metagenomic DNA sequencing of first round PCR amplicons produced from the V4 region from the 16S ribosomal RNA (rRNA) gene in bacteria:  Template DNA derived from root of a seedling whose seed was collected under mother tree 4 and grown in soil containing an inoculum of forest soil from under mother tree M3
design: Immediately after soil removal by washing with water, seedling roots were placed in sterile plastic tubes and flash frozen in liquid nitrogen.  They were then stored at -80 degrees C until shipped in a liquid nitrogen dry shipping container to the Marden Lab Penn State where they were transferred without thawing to a -80 freezer for storage.  In the Marden Lab, roots were ground under liquid nitrogen to a powder in a porcelain mortar and pestle.  The powder was transferred under liquid nitrogen to a sterile plastic tube and then without thawing to a -80 freezer for storage.  An aliquote of about 50 mg of sample powder was transferred without thawing to a sterile nuclease free 1.7 ml microcentrifuge tubes.  DNA in the sample aliquote was isolated using the Machery-Nagel NucleoSpin Plant II Mini Kit and subsequently cleaned using the Machery-Nagel NucleoSpin gDNA Clean-up Kit.  Isolated DNA was stored at -20 degrees C.  This DNA was used to produce amplicons from bacterial 16S V4 in a simplex first round of PCR using Phusion Hot Start II High Fidelity Polymerase and primer annealing at 50C.  Primers were 515V-v1 (5-GTGCCAGCMGCCGCGGTAA-3) and 805R-v1 (5-GGACTACHVGGGTWTCTAAT-3) that had the respective Illumina Nextera-style overhang adapter attached to the 5end.  Amplicons were cleaned using Agencourt AMPure XP beads cleaned amplicons were examined on an Agilent 2100 Bioanalyzer to revealed only one peak of about 375 bp as expected.  First round amplicons were supplied to the Penn State Genomics Core Facility who performed second round PCR (Kapa HiFi HotStart PCR Kit) to add and sequencing.  Second round PCR used the Kapa HiFi HotStart PCR Kit (KAPA Biosystems) to add Illumina sequencing adapters and Nextera duel indices.  Second round amplicons were purified and normalized for equal molarity using SequalPrep Normalization Plates sequenced on an Illumina MiSeq running the MiSeq Reagent Kit v2, 500 cycles with PhiX at 5%.