where we can assess impact on reproducibility of the same parameters discussed so far (expression level and probe place- ment) while disregarding platform differences. We hypothe- sized that reproducibility within each platform (for those genes with multiple probes predicted to target them) would show the same trends as reproducibility across platforms. We ﬁrst found that within-platform ‘reproducibility’ was substantially lower on the Illumina array than for Affymetrix or between-platform reproducibility (9.5% of 4312 correlations over 0.8 on Illu- mina; 27.2% of 30 384 comparisons for Affymetrix, compared with 24% between platforms; see Supplementary Data for details). We interpret this as indicating that the Affymetrix array contains more probe sets that are ‘truly redundant’, at least as reﬂected in our tissue samples. We also conﬁrmed that the expression level and probe location appear to play a similar role in reproducibility within platforms as they do between platforms, i.e. two probes targeting the same gene within a platform were more likely to yield concordant results if they exhibited stronger expression and were targeting nearby sites in the genome (see Supplementary Data for details). ‘Unexplained’ cases of disagreement There are probes which, based on dilution effect, location and expression level criteria, would be predicted to yield repro- ducible results, but do not. This suggests that other factors are inﬂuencing the results, and further examination of these cases is warranted. To examine this in more detail, we sought to identify provisionally ‘unexplained’ cases of disagreement by ﬁltering the full set of results, using partly arbitrary criteria. We removed genes for which the 30 ends of the probes were located further apart than 100 bases (similar in size to the average human exon) between the two platforms. We then ﬁltered out probes which were expressed at low levels on both platforms (medians below the 25th percentile). Finally, we removed pairs which showed good agreement across plat- forms (as these need no further explaining), setting a maxi- mum correlation threshold of 0.5 (close to that which maintained an FDR of 0.05), and also required that at least one of the probes show a strong dilution effect (again using the threshold of 0.5, but as a lower limit). This leaves a set of 940 pairs of probes for further study, or 3% of all compar- isons. We do not suggest this set represents all the disagree- ments, just a subset of harder-to-explain disagreements. By far the most striking disagreements among the selected probes are those exhibiting strong negative correlations across platforms, i.e. both platforms indicate a strong dilution effect for the gene, but in the opposite direction. There are 41 pairs of probes in this set which show cross-platform rank correlations of < 0.5. These probes appear wholly unremarkable based on the parameters we have focused on (expression level and loca- tion), compared with the 899 other probe pairs (the complete list of the 940 probe pairs are provided as Supplementary Data). Most disagreements are more subtle. If one focuses on probe pairs that show cross-platform correlations of <0.2, the number of selected probes is reduced by about a factor of two (that is to say, quite a few of the ‘unexplained disagreements’ involve marginal cases with correlations between 0.2 and 0.5). To attempt to further explain the 940 cases, we ﬁrst hypothesized that despite having similar genomic locations of the centers of the targeted sequences, there might be larger Nucleic Acids Research, 2005, Vol. 33, No. 18 5921 differences in the sequences assayed on the two platforms. We considered this plausible because Affymetrix probe sets assay more sequence and often include probes spread fairly widely (a median of 481 genomic bases from the 50 end of the 50-most probe to the 30 end of the 30-most probe) compared with the Illumina platform, which use a single 50 bp probe that almost always maps to 50 contiguous bases in our analysis. This could lead to different populations of transcripts being assayed in some cases. However, there was no signiﬁcant enrichment in alignments shorter or longer than the median in the set of 940 pairs (P > 0.05, Fisher’s exact test), suggest- ing that there is no overall pattern of alignment statistics that can explain the relatively anomalous behavior of these 940 pairs. Next, we counted the number of different transcripts pre- dicted to be hybridized by each probe (assuming for the moment that all RNAs are equally likely to be detected, regardless of 30 location of the probe). If these values are different on the two platforms, then agreement may be lower if the predicted transcripts are indeed present in the tissues we studied. However, there was no overall difference between Affymetrix and Illumina in the number of transcripts assayed among the set of 940 (P 0.3, paired t-test). A potential remaining source of ‘disagreement’ could be differential cross-hybridization. Similar to transcript speciﬁ- city, cross-hybridization is difﬁcult to analyze computation- ally because it could involve platform-speciﬁc differences that might not be reﬂected in the probe sequences alone (e.g. syn- thesis efﬁciency on the Affymetrix platform, or the effect of the probe identiﬁcation sequences and linker for Illumina probes), and other unknowns such as the impact of highly expressed but weakly cross-hybridizing transcripts. There are 250 probes on each platform that have very high poten- tial for cross-hybridization based on our sequence analysis (see Materials and Methods). If anything these are slightly under- represented among the 940 strongest disagreements (Fisher’s exact test, P ¼ 0.036, Illumina; 0.07, Affymetrix). We also note that on the Affymetrix platform, where the manufacturer ‘ﬂags’ probe sets with the potential for various types of non- speciﬁcity, there is no difference in the proportion of ﬂagged probe sets among the 940 compared with the rest of the probes (38 versus 37%). We performed a similar analysis for the within-platform analyses. Within-platform reproducibility showed many fewer hard-to-explain failures of reproducibility. For the Illumina platform, application of the same ﬁlter that yielded 940 pairs of probes (2.6% of the total) for the cross-platform compari- son yielded 10 pairs of probes (0.2%), while for Affymetrix it yielded 103 pairs (0.3%). Interestingly, 3 of these Illumina probes and 37 of the Affymetrix probes appear in the list of 940 probes involved in poor ‘unexplained’ cross-platform comparisons. This enrichment is highly signiﬁcant (P ¼ 0.0039 and P < 10 15 for Illumina and Affymetrix, respec- tively). Our interpretation of this ﬁnding is that these probes are somehow inherently ‘poorly behaved’ and we predict that they will not yield biologically useful results. DISCUSSION Our main conclusion from this study is that the Affymetrix and Illumina platforms yield highly comparable data, especially