amplicon sequncing of soil fungi in a temperate agroforestry system
Primer pair ITS1-F_KYO2 (5-TAGAGGAAGTAAAAGTCGTAA-3) (Toju et al. 2012) / ITS86R (5-TTCAAAGATTCGATGATTCA-3) (Vancov & Keen 2009); Thermocycling conditions were as follows: initial denaturation (95C for 2 min), 3 touch-up cycles of denaturation (95C for 20 sec), annealing (50C for 30 sec), and elongation (68C for 60 sec), 25 cycles of denaturation (95C for 20 sec), annealing (58C for 30 sec), and elongation (68C for 60 sec), and final elongation (68C for 5 min). Following thermocycling, successful amplification was confirmed by visualizing 2 L of the PCR product on 1.7 % (w/v) agarose gels stained with SYBR Green I solution (Thermo Fisher Scientific GmbH, Dreieich, Germany). Following thermocycling, successful amplification was confirmed by visualizing 2 L of the PCR product on 1.7 % (w/v) agarose gels stained with SYBR Green I solution (Thermo Fisher Scientific GmbH, Dreieich, Germany). Gel electrophoresis was performed at 4.6 V cm2 for 60 min. Libraries were shipped to the facilities of LGC Genomics (Berlin, Germany) for a second amplification step using standard i7- and i5- sequencing adaptors. The second PCR was performed in 20 L reaction volume containing 1 MyTaq buffer (Bioline GmbH, Luckenwalde, Germany), 15 pmol of each forward and reverse i7- and i5- sequencing adaptors, 2 l of BioStabII PCR Enhancer (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany), and 0.075 u L1 MyTaq DNA polymerase  (Bioline GmbH, Luckenwalde, Germany).The thermocycling conditions were as follows: initial denaturation (96 C for 1 min), 3 touch-up cycles of denaturation (96 C for 15 s), annealing (50 C for 30 s), and elongation (68 C for 90 s) followed by 7 cycles of denaturation (96 C for 15 s), annealing (58 C for 30 s), and elongation (68 C for 90 s), and final elongation (70 C for 2 min). DNA quantity was assessed on agarose gels and indexed sequencing libraries were pooled. The multiplexed libraries were sequenced using on an Illumina MiSeq unsing V3 chemistry (2  300 bp) (Illumina, Inc., San Diego, CA, USA) at LGC Genomics, Berlin, Germany.