Sequence dependence of cross-hybridization on short oligo microarrays

One of the critical problems in the short oligo microarray technology is how to deal with cross-hybridization that produces spurious data. Little is known about the details of cross-hybridization effect at molecular level. Here, we report a free energy analysis of cross-hybridization on short oligo microarrays using data from a spike-in study. Our analysis revealed that cross-hybridization on the arrays is mostly caused by oligo fragments with a run of 10–16 nt complementary to the probes. Mismatches were estimated to be energetically much more costly in cross-hybridization than that in gene-specific hybridization, implying that the sources of cross-hybridization must be very different between a PM–MM probe pair. Consequently, it is unreliable to use MM probe signal to track cross-hybridizing signal on a corresponding PM probe. Our results also showed that the oligo fragments tend to bind to the 5′ ends of the probes, and are rarely seen at the 3′ ends. These results are useful for microarray design and data analysis.