Computational protocol: Volatile organic compounds in truffle (Tuber magnatum Pico): comparison of samples from different regions of Italy and from different seasons

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Protocol publication

[…] To identify relationships among the samples (Alba, A; San Miniato winter, SM; San Miniato summer SM*) based on data obtained from PTR-TOF-MS, multiple factorial analyses (MFA) was used. MFA was performed in two steps. Firstly, a principal component analysis (PCA) was computed on each data set, which was then “normalized” by dividing all its elements, by the square root of the first eigenvalue obtained from of its PCA. Then, the normalized data sets are merged to form a single matrix and a global PCA is performed on this matrix. The individual data sets are then projected onto the global analysis to analyze communalities and discrepancies. Volatile compounds significantly contributing to MFA dimensions were used to explain differences among truffles (normal law adjustment test on compounds correlation coefficients, α = 0.05). A hierarchical clustering on principal components (HCPC) was performed to confirm the product groups observed graphically. Heat maps method were used for visualizing complex data sets organized as matrices. A heat map does two things to a matrix. First, it reorders the rows and columns so that rows (and columns) with similar profiles are closer to one another, rendering them to be more visible to the eye. Second, each entry in the data matrix is displayed as a color, making it possible to view the patterns graphically. The dendrograms were created using correlation-based distances and the Ward method of agglomeration was used in the present analysis. All computations were performed with R 3.0.3 language and environment and R packages FactoMineR, and gplots were used. [...] Total genomic DNA was extracted from a sample named as “Marcia” using the CTAB extraction method with minor modifications. Next, the ITS region was amplified with the ITS5/ITS6 pair of primers using a Biorad MyCycler system in a 25 μl of mixture solution containing 100 ng of DNA from fruiting bodies. Amplification was performed using the following protocol to get each sequence. PCR amplification with the pair of primers ITS5/ITS6 was carried using the method described in reference. Electrophoresis on agarose gel (2 μl of PCR mixture, 2% agarose gel) with ethidium bromide staining confirmed that the PCR products were of the predicted size ITS5/ITS6 (600–650 bp). The amplicons were purified trough Wizard SV Gel and PCR Clean-Up System Kit (Promega) and then sequenced (BMR Genomics, Padova Italy) to get their relative sequences.The sequences thus obtained were inserted into a multiple sequence alignment program, using the MUSCLE alignment algorithm. A neighbor-joining tree was constructed based on maximum likelihood (PhyML) using the web resource available on the website (, an high performance platform designed to perform phylogenetic analysis based on a multiple alignment. The phylogenetic tree constructed using this data helped define the species to which the summer fruiting bodies belong. […]

Pipeline specifications

Software tools FactoMineR, gplots, MUSCLE, PhyML,
Applications Miscellaneous, Phylogenetics, Nucleotide sequence alignment
Diseases Pilomatrixoma