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

Hedda Høvik,Tsute Chen

doi:10.1186/1471-2105-11-82

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.

Highlights

Current 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
We have applied the described pipeline to design genomic tiling array probe sets for several microbial genomes that are currently available in the NCBI database [35]
The window size was determined based on the genome size and the number of probe spots on the microarray

Summary

Introduction

Current 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. Maskless array synthesis (MAS) technology [2] makes it possible to synthesize oligonucleotides of any sequence This great capacity can be used for designing probe sequences covering an entire small genome or chromosome or a continuous portion of larger genomes. They have been used to study whole genome or chromosome transcription for various organisms including humans [3,4,5,6] For small genomes such as those of prokaryotes, the capacity of the highdensity tiling arrays often allows for studies of the comprehensive transcriptome [7,8,9]

Objectives

Results

Discussion

Conclusion