Selecting of oligonucleotide probes for protein coding sequences

Motivation: Large arrays of oligonucleotide probes have become popular tools for analyzing RNA expression. However to date most oligo collections contain poorly validated sequences or are biased toward untranslated regions (UTRs). Here we present a strategy for picking oligos for microarrays that focus on a design universe consisting exclusively of protein coding regions. We describe the constraints in oligo design that are imposed by this strategy, as well as a software tool that allows the strategy to be applied broadly. Result: In this work we sequentially apply a variety of sim-ple filters to candidate sequences for oligo probes. The pri-mary filter is a rejection of probes that contain contiguous identity with any other sequence in the sample universe that exceeds a pre-established threshold length. We find that rejection of oligos that contain 15 bases of perfect match with other sequences in the design universe is a fea-sible strategy for oligo selection for probe arrays designed to interrogate mammalian RNA populations. Filters to re-move sequences with low complexity and predicted poor probe accessibility narrow the candidate probe space only slightly. Rejection based on global sequence alignment is performed as a secondary, rather than primary, test, lead-ing to an algorithm that is computationally efficient. Splice isoforms pose unique challenges and we find that isoform prevalence will for the most part have to be determined by analysis of the patterns of hybridization of partially redun-dant oligonucleotides. Availability: The oligo design program OligoPicker and its source code are freely available at our website