Ophir et al. BMC Genomics 2010, 11:269 http://www.biomedcentral.com/1471-2164/11/269 Page 5 of 10 Figure 2 SFP discovery and validation at the Unigene level. Examples for genes MU16815 and MU17949 are shown. The graph shows that the signal ratio of the putative SFP is stronger than all other probe sets of the same gene. The signal ratio (y axis) is plotted against the probe position relative to the 5' end of the UniGene contig (x axis). The red dots represent the probes that are predicted to contain SFPs based on statistical analysis. The signal ratio consists of 'Dulce' signal to PI414723 signal, and therefore a positive log signal ratio represents a PI414723 SFP, i.e., 'Dulce' better matches the probe. A negative log signal ratio represents a 'Dulce' SFP. The variation in 'Dulce' and PI414723 SFPs (corresponding to the red dots in the top part of the figure) is revealed by sequencing, illustrated in the bottom part of the graph. to the SFPs. Nevertheless, these differences could still be used as markers. In this work we tried to exclude the pos- sibility of minor frequency alleles (MFAs). For example, focusing on a specific locus, in one parent the allele may be T in its two biological replicates (pools) whereas in the other parent gDNA pools, the T allele might make up only 80% of the pool and G might make up the other 20%. This might affect the hybridization signal, but it would not be observable at the sequence level. By showing that the inter-population variation is greater than the intra- population variation, we exclude this possibility (Addi- tional file 2). Luo et al. [36], using labeled cDNA and Affymetrix array in barley, suggested that many of the SFPs that do not show any differences in direct sequenc- ing are a result of cis-regulatory expression regulators. This explanation is not relevant to our study as we are using genomic DNA for labeling. As only partial tran- scriptome data exist for melon, the ability to discover SFPs and to eliminate cross-hybridizations and gene duplications is limited. Divergence of exon-intron bound- aries and alternative splicing of cDNA that are printed on the array and the labeled genomic DNA could also con- tribute to the high FDR that is found by sequencing. Another explanation for the discrepancy in FDR can be the power of the statistical test, which is might be too low given the number of replicates in the different experi- mental systems, We do not believe that this is the reason for the high FDR as we used the empirical Bayesian model that can repair low power in limma [28]). One can improve the discovery rate by ignoring all multiple SFPs (four or more), which can be considered copy number variations (Figure 2). It is also possible that by using this type of long probe array, we lose a portion of the varia- tion; however in doing so, we gain better coverage of the