Abstract
BackgroundHigh-density tiling microarrays are a powerful tool for the characterization of complete genomes. The two major computational challenges associated with custom-made arrays are design and analysis. Firstly, several genome dependent variables, such as the genome's complexity and sequence composition, need to be considered in the design to ensure a high quality microarray. Secondly, since tiling projects today very often exceed the limits of conventional array-experiments, researchers cannot use established computer tools designed for commercial arrays, and instead have to redesign previous methods or create novel tools.Principal FindingsHere we describe the multiple aspects involved in the design of tiling arrays for transcriptome analysis and detail the normalisation and analysis procedures for such microarrays. We introduce a novel design method to make two 280,000 feature microarrays covering the entire genome of the bacterial species Escherichia coli and Neisseria meningitidis, respectively, as well as the use of multiple copies of control probe-sets on tiling microarrays. Furthermore, a novel normalisation and background estimation procedure for tiling arrays is presented along with a method for array analysis focused on detection of short transcripts. The design, normalisation and analysis methods have been applied in various experiments and several of the detected novel short transcripts have been biologically confirmed by Northern blot tests.ConclusionsTiling-arrays are becoming increasingly applicable in genomic research, but researchers still lack both the tools for custom design of arrays, as well as the systems and procedures for analysis of the vast amount of data resulting from such experiments. We believe that the methods described herein will be a useful contribution and resource for researchers designing and analysing custom tiling arrays for both bacteria and higher organisms.
Highlights
The availability of affordable custom-made expression arrays is increasing, and the feature number on oligonucleotide microarrays has increased remarkably during the last few years
We believe that the methods described will be a useful contribution and resource for researchers designing and analysing custom tiling arrays for both bacteria and higher organisms
Present analysis methods for microarrays are mainly focused on known coding regions [8,10], and researchers soon run into problems when trying to analyse signals from intergenic regions or un-annotated genomes, because of the difficulty in defining consistently expressed segments of the genome without the aid of an annotation. These problems can be addressed by applying the methods presented here, and the annotation-independent analysis method can be applied to any tiling array project, regardless of whether the investigated regions are coding or non-coding, and without the need of any genomic annotation or training set. In this manuscript we present a novel design method for tiling arrays, here targeting prokaryotic genomes, but applicable to eukaryotic genomes as well
Summary
The availability of affordable custom-made expression arrays is increasing, and the feature number on oligonucleotide microarrays has increased remarkably during the last few years. Recent reports show that annotated genes tend to contain methylation sites with biased distribution towards the 39 end. This bias in the expressed gene indicate that methylation might interfere with transcription initiation and termination [3,4]. To address this problem, new microarray approaches that enable mapping of the total genome have emerged [5]. Several genome dependent variables, such as the genome’s complexity and sequence composition, need to be considered in the design to ensure a high quality microarray. Since tiling projects today very often exceed the limits of conventional arrayexperiments, researchers cannot use established computer tools designed for commercial arrays, and instead have to redesign previous methods or create novel tools
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