title: Glomus amplicon pyrosequencing
design: Potential single copy marker sequences were amplified using DreamTaq DNA polymerase (Fermentas) using primers listed in Table S3, with suitable adapter, key and MID sequences added. DNA from the strain G. irregulare DAOM 197198 was extracted as described in (22) The reaction was performed in 50 µl containing 1 ng DNA, 1.25 U Taq polymerase (Fermentas), 0.2 mM dNTPs, 0.4 µM of each primer and the PCR buffer. PCR was carried out for 40 cycles, i.e. 94°C for 30 sec, Ta for 30 sec (see Table S3 for annealing temperature per primer pair) and 72°C for 1 min, which were preceded by an initial 3 min denaturation at 95°C and followed by a 10 min hold at 72°C, on a Mastercycler EPgradient S (Eppendorf). The PCR product was checked on an electrophoresis gel to ensure successful amplification of the gene, and then purified using a MinElute PCR Purification Kit (Qiagen) according to manufacturer’s instructions. These purified samples were pooled by molecular weight and sent to the Genome Québec Innovation Centre (McGill University, Montréal) for pyrosequencing using the GS FLX Titanium emPCR kit (Roche 454 Life Science) with lib-L chemistry in 1/8 run.