Dynamic probe selection for studying microbial transcriptome with high-density genomic tiling microarrays

Abstract

BackgroundCurrent commercial high-density oligonucleotide microarrays can hold millions of probe spots on a single microscopic glass slide and are ideal for studying the transcriptome of microbial genomes using a tiling probe design. This paper describes a comprehensive computational pipeline implemented specifically for designing tiling probe sets to study microbial transcriptome profiles.ResultsThe pipeline identifies every possible probe sequence from both forward and reverse-complement strands of all DNA sequences in the target genome including circular or linear chromosomes and plasmids. Final probe sequence lengths are adjusted based on the maximal oligonucleotide synthesis cycles and best isothermality allowed. Optimal probes are then selected in two stages - sequential and gap-filling. In the sequential stage, probes are selected from sequence windows tiled alongside the genome. In the gap-filling stage, additional probes are selected from the largest gaps between adjacent probes that have already been selected, until a predefined number of probes is reached. Selection of the highest quality probe within each window and gap is based on five criteria: sequence uniqueness, probe self-annealing, melting temperature, oligonucleotide length, and probe position.ConclusionsThe probe selection pipeline evaluates global and local probe sequence properties and selects a set of probes dynamically and evenly distributed along the target genome. Unique to other similar methods, an exact number of non-redundant probes can be designed to utilize all the available probe spots on any chosen microarray platform. The pipeline can be applied to microbial genomes when designing high-density tiling arrays for comparative genomics, ChIP chip, gene expression and comprehensive transcriptome studies.

RESEARCH PAPER A stable density approach to probe selection for a custom aCGH design

“…The solutions proposed in the previous works usually attempted to overcome this issue by either ignoring these gaps (Lipson et al, 2007) or by using lower quality probes (Schliep and Krause, 2008;Thomassen et al, 2009;Hovik and Chen, 2010).…”

“…Consequently, the likelihood of rearrangements in such regions is greater than in other genomic locations (Stankiewicz and Lupski, 2010). It is becoming extremely important to pay a special attention on poorly covered regions (Hovik and Chen, 2010). Moreover, most of the eukaryotic non-protein coding sequences are low complexity sequences, such as repetitive elements (Ahnert et al, 2008).…”

“…An example of the mixed method is presented in (Hovik and Chen, 2010). The authors propose a twostage approach to the problem of probe selection.…”

Roseomics: a blank slate

“…Due to the reproducibility, low cost, high sensitivity and high specificity of tiled arrays (50), development of virus-specific arrays for HHV-6A -6B and -7 should be prioritized. The production and use of tiled arrays is a standard commodity at most institutions and offers a unified platform for transcript characterization and quantification.…”

Microarray oligonucleotide probe designer: a Web service

“…The central challenge is then the selection of the specific oligonucleotides to be placed on a genomic selection array 4, 9. Although there are a number of algorithms for designing tiling arrays for genome-wide transcriptome or ChIP-seq experiments 15–18. (see review in 19), we still lack easily accessible open-sources tools for building genomic selection arrays.…”