Extracting more signal at high intensities in oligonucleotide arrays

Motivation: The majority of work in the analysis of microarray data has focused on improving the signal to noise ratio in the low concentration region. However, improvements are also possible at the opposite extreme: at high concentrations. This problem appears to have been neglected for two reasons: first, it is counter-intuitive to expect problems in what appears to be the cleanest region and second, the data necessary to identify and develop improvements has not been widely available. Fortunately, the situation has changed and precise calibration data produced by Affymetrix which enables the analysis of signals over a broad range of concentrations. Using this data, we address the question: “Where on the concentration/intensity scale are we able (or unable) to detect fold changes of 2 and greater? ” Results: First, we empirically demonstrate that while current techniques are capable of detecting changes in the low to middle concentration range, the situation is quite different for high concentrations. Specifically, in this regime, expression changes as large as 4 fold are severely compressed, and changes of 2 are often undetectable. However, a feature of GeneChips is that transcripts are probed by a set of sequence fragments having a wide range of affinities. The key to improving the accuracy in a given intensity range, is choosing those probes that are most responsive in that range. Utilizing this property and borrowing ideas from the theory of local linear embeddings (LLE) we present a new approach capable of reducing the compression at high intensity. Availability: Program source code will be sent electronically upon request. Contact: felix@funes.rockefeller.edu; soccin@rockefeller.edu; marcelo@zahir.rockefeller.edu Introduction High-density oligonucleotide arrays manufactured by Affymetrix are currently among the most sensitive and reliable microarray technology (Chee et al., 1996; Lipshutz et al., 1999) available. Based on a photolithographic oligonucleotide deposition process, labeled and amplified mRNA transcripts are probed by 28-40 short DNA sequence each 25 bases long. The probes are preferentially picked near the 3’ end of the mRNA sequence, because of the limited efficiencies of reverse transcription enzymes. In addition, the probes come in two varieties: half are perfect matches (PM ) identical to templates found in databases, and the other half are single mismatches (MM ), carrying a single base substitution in the middle (13th) base of the sequence. MM probes were introduced to serve as controls for non-specific hybridization, and is was thought that the actual signal (the target’s mRNA concentration) would be proportional to the difference of match versus mismatch (PM-MM ). We shall confirm below that as a first approximation, this subtraction procedure is the most reasonable choice, leading to remarkably good results in the low to mid concentration range. However, we shall show that for high concentrations, the signal tends to be suppressed when using differentials. This result is likely to sound surprising and counter-intuitive to many, which we believe is rooted in the following widespread interpretation of hybridization data. Namely, when examining a typical plot of the data from two replicated conditions (Figure 1a) most would immediately focus on the low intensity region, and observe how noisy this regime appears to be in comparison to the much cleaner highintensity range. However, matters turn out to be more complex and we shall argue this view is at least partly incorrect and often misleading. Turning to a comparison of two different conditions (Figure 1b), we notice that the noise envelope is essentially unchanged, and that real changes appear as points lying