title: Development and validation of an Haemophilus influenzae supragenome hybridization (SGH) array for transcriptomic analyses.
abstract: We previously carried out the design and testing of a custom-built Haemophilus influenzae supragenome hybridization (SGH) array that contains probe sequences to 2,890 gene clusters identified by whole genome sequencing of 24 strains of H. influenzae. The array was originally designed as a tool to interrogate the gene content of large numbers of clinical isolates without the need for sequencing, however, the data obtained is quantitative and is thus suitable for transcriptomic analyses. In the current study RNA was extracted from H. influenzae strain CZ4126/02 (which was not included in the design of the array) converted to cDNA, and labelled and hybridized to the SGH arrays to assess the quality and reproducibility of data obtained from these custom-designed chips to serve as a tool for transcriptomics. Three types of experimental replicates were analyzed with all showing very high degrees of correlation, thus validating both the array and the methods used for RNA profiling. A custom filtering pipeline for two-condition unpaired data using five metrics was developed to minimize variability within replicates and to maximize the identification of the most significant true transcriptional differences between two samples. These methods can be extended to transcriptional analysis of other bacterial species utilizing supragenome-based arrays.
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania, United States of America; Department of Microbiology and Immunology, Drexel University College of Medicine, Allegheny Campus, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Janto
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania, United States of America; Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania, United States of America.
authors.forename: N Luisa
authors.initials: NL
authors.lastname: Hiller
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Rory A
authors.initials: RA
authors.lastname: Eutsey
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Dahlgren
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Joshua P
authors.initials: JP
authors.lastname: Earl
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Evan
authors.initials: E
authors.lastname: Powell
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Azad
authors.initials: A
authors.lastname: Ahmed
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania, United States of America; Department of Microbiology and Immunology, Drexel University College of Medicine, Allegheny Campus, Pittsburgh, Pennsylvania, United States of America; Department of Otolaryngology Head and Neck Surgery, Drexel University College of Medicine, Allegheny Campus, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Fen Z
authors.initials: FZ
authors.lastname: Hu
authors.affiliation: Center for Genomic Sciences, Allegheny-Singer Research Institute, Pittsburgh, Pennsylvania, United States of America; Department of Microbiology and Immunology, Drexel University College of Medicine, Allegheny Campus, Pittsburgh, Pennsylvania, United States of America; Department of Otolaryngology Head and Neck Surgery, Drexel University College of Medicine, Allegheny Campus, Pittsburgh, Pennsylvania, United States of America.
authors.forename: Garth D
authors.initials: GD
authors.lastname: Ehrlich
mesh_terms: D020869:Gene Expression Profiling; D016680:Genome, Bacterial; D023281:Genomics / Q000379:methods; D006193:Haemophilus influenzae / Q000235:genetics*; D006801:Humans; D009693:Nucleic Acid Hybridization; D015203:Reproducibility of Results; D059467:Transcriptome