In total, 74 fungal species were probed via the fungal amplicon mixes. The PCR product that was amplified from the ITS region of Arabidopsis thaliana
was added to all amplicon mixes (at a concentration of 5 ng/μl) as a positive hybridisation control. To test the possible use of this custom phylochip for describing ECM community composition Peptide 17 molecular weight in environmental samples, 10 μl of the PCR product that was amplified from the bulked ECM root tips of beech and spruce was used (spiked with the amplicon of Arabidopsis thaliana). Six technical replicates were carried out for each sample (three block replications per slide × two slides per sample). The results of the cross-hybridisation test are outlined in Figure 1. The ITS-based cladogram was constructed for all tested fungal species using the default setting of the MEGAN software (version 3.0.2., [42]). Array evaluation Prior to further analyses,
spots exhibiting poor quality (for example, as a result of the presence of dust) were flagged and excluded from the analyses. Hybridisation quality was surveyed using the positive (oligonucleotides of Arabidopsis thaliana) and negative controls (five oligonucleotides for the Glomeromycota (non-ECM species) and the one spot spotted with only hybridisation buffer) of each array. Data of the array were further used when (i) signal intensity values of the positive controls were within the group of oligonucleotides that showed the highest signal intensity values Olopatadine and (ii) Volasertib price the mean signal intensity value of the negative controls were a maximal 1.5% of the signal intensity with the highest value. Individual spots were considered to be positive (species present in the sample) if their signal intensity showed a value that was five-fold higher than the averaged intensity value for all of the negative controls. Additionally, at least four of the six replicates per spot were required to generate a significant positive hybridisation. The threshold factor was fixed to five-fold after evaluation of the results of the arrays that were hybridised with the
known amplicon mixes derived from sporocarp tissues (see “”Sporocarp collection”" and “”Specificity of oligonucleotides”"). Using a threshold factor of “”5″” defined the minimal 90% of all species in the amplicon mixes as positive and filtered most false-positives (cross-hybridisation). Acknowledgements MR is supported by a Marie Curie PhD scholarship within the framework of the TraceAM programme. The array approach was partly funded by INRA, the European projects TraceAM and ENERGYPOPLAR, the European Network of GSK621 cost Excellence EVOLTREE, and the Typstat project (GIP ECOFOR). We would like to thank Dr. Melanie Jones (University of British Columbia Okanagan) for her critical reading of the manuscript and helpful comments. We also thank Christine Delaruelle (INRA-Nancy) for her technical assistance with the ITS sequencing.
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