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Each genotype was represented by eight biological replicates in a dye-swap loop design.
Genes were considered to be differentially expressed if they exhibited a 2-fold perturbation in gene expression magnitude in each of the three biological replicates, in a statistically significant manner (P<0.05).
PCA of this subset of SEM demonstrated improved grouping of the PT and PP biological replicates in a 3 principal component space (Fig 4B).
Four biological replicates were performed for each experimental condition and dye-sample coupling was swapped between biological replicates in a balanced block design.
PCA of the same dataset resulted in an approximate grouping of the PT and PP biological replicates in a 3 principal component space (Fig 4A; Additional file 2).
RT-qPCR reactions were performed in technical triplicates from at least two biological replicates in a StepOnePlus Real Time PCR System (Applied Biosystems).
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Experiments were carried out using four biological replicates in an Applied Biosystems 7500 equipment.
By clustering normalized expression values from different biological replicates in all data sets, we generated an overall "pathovivogram" that includes 276 genes in ten coexpression clusters, CCs (Fig. 2, Fig. S2, and Table S1).
Our finding further emphasises the need for sufficient technical and biological replicates in all microarray experiments.
For the two assays for which only qualitative data were available (complex colony morphology and complex mat formation), we performed a Kendall's tau rank correlation test on the qualitative scores across all strains for a single biological replicate in a pairwise fashion.
The use of the three biological replicates in DESeq2 allowed a number of statistical analyses to be performed.
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