Unknown,Transcriptomics,Genomics,Proteomics

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Supervised Lowess normalization of comparative genome hybridization data: application to lactococcal strain comparisons.

ABSTRACT: Background Array-based comparative genome hybridization (aCGH) is commonly used to determine the genomic content of bacterial strains. In aCGH data, systematic errors are comparable to those occurring in transcriptome data. However, especially for microbes, an additional source of variation exists: differences in hybridization due to gene sequence divergence between the strains hybridized. Current normalization methods do not take this source of variation into consideration. Results We present Supervised Lowess, or S-Lowess, an application of the subset Lowess normalization method that does take difference in genomic content into account. The performance of S-Lowess was assessed on aCGH experiments in which differentially labeled genomic DNA fragments of Lactococcus lactis IL1403 and L. lactis MG1363 strains were hybridized to IL1403 microarrays. Since both genome sequences are known (they have only on average 85 % sequence identity), the success rate in detecting deletions in the MG1363 genome of different aCGH normalization methods can be compared. S-Lowess detects 97% of the deletions, whereas other aCGH normalization methods detect up to only 60% of the deletions. Conclusions S-Lowess removes systematic errors from dual-dye aCGH data in two steps: (i) determination of likely homologous genes (LHG); and (ii) estimation of correction factors for systematic errors from spots of LHG and subset Lowess normalization of the remaining spots using these correction factors. It is implemented in a user-friendly web-tool accessible from http://bioinformatics.biol.rug.nl/websoftware/s-lowess. We demonstrate that it outperforms existing normalization methods and maximizes the number of detectable genomic deletions or duplications from microbial aCGH data. Keywords: comparative genome hybridisation In this study, 4 aCGH comparisons (slides) between L. lactis MG1363 and L. lactis IL1403 were performed (including dye swap with biological replicates; see also supplementary materials). The resulting aCGH slide signals were normalized using the different 'likely homologous gene' (LHG) sets (see above) yielding ratios of signals of labelled DNA of MG1363 over those of IL1403. A maximum of 8 ratios per amplicon (gene) were obtained for the 4 hybridized slides (each with 2 replicate spots per amplicon). Only genes with at least 5 measurements were used in this study. The normalization methods evaluated in this study are: a) no normalization, b) total signal normalization, c) grid-based Lowess (implemented in PreP; f = 0.7) [GarcM-CM--a de la Nava, J et al. 2003. Bioinformatics 19:2328-2329], and d) S-Lowess using different subsets of conserved lactococcal genes (for details see above). Results with the MANOR R package (spatial normalization; standard parameters) [Neuvial P, et al. 2006. BMC Bioinformatics 7:264; Liva S, et al. 2006. Nucleic Acids Res 34:W477-W481] are shown in our supplementary materials.

ORGANISM(S): Lactococcus lactis subsp. cremoris MG1363

SUBMITTER: Sacha van Hijum

PROVIDER: E-GEOD-9039 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Supervised Lowess normalization of comparative genome hybridization data--application to lactococcal strain comparisons.

van Hijum Sacha A F T SA Baerends Richard J S RJ Zomer Aldert L AL Karsens Harma A HA Martin-Requena Victoria V Trelles Oswaldo O Kok Jan J Kuipers Oscar P OP

BMC bioinformatics 20080211

<h4>Background</h4>Array-based comparative genome hybridization (aCGH) is commonly used to determine the genomic content of bacterial strains. Since prokaryotes in general have less conserved genome sequences than eukaryotes, sequence divergences between the genes in the genomes used for an aCGH experiment obstruct determination of genome variations (e.g. deletions). Current normalization methods do not take into consideration sequence divergence between target and microarray features and theref ...[more]

PMID: 18267014

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Project description:Although immune mechanisms have been described for Peyer’s patch function, in discriminating food nutrients and commensal microflora from pathogenic challenge, an understanding of gene expression patterns associated with normal intestinal homeostasis is lacking. The effects of commensal bacterial colonization on global gene expression in the small intestine of germ-free mice have been described (1, 2). However, no studies have compared expression patterns of various healthy lymphoid tissues in conventionally-raised pigs or other species. The objective of this study was to investigate the pattern of normal gene expression in jejunal Peyer’s patches (JPP) of healthy pigs. We hypothesized that gene expression of intact jejunal Peyer's patch is characterized by genes associated with absorption and secretory functions as well as genes associated with GALT-specific immune functions. Eight hybridizations were performed, with dye swaps of JPP mRNA from four juvenile pigs. Each hybridization compared JPP from an individual pig to the jejunal mesenteric lymph node reference sample, which was pooled from all juvenile pigs in the study. However, two of the hybridizations resulted in poor quality data, and were excluded from further analysis. Normalization, hierarchical clustering, data visualization, and statistical analysis were performed by GeneSpring version 6.2 (Silicon Genetics, Redwood, CA). GeneSpring standardized the intensities for each spot by subtracting the local background, and then normalized globally by locally weighted linear regression (Lowess). A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. Spots with a flag value of “0” were excluded. The intensity of negative control DNA-containing spots was used to additionally exclude low intensity data. Negative controls include the gene for Arabidopsis thaliana homeodomain-like protein (AY054571); 200 ng/microl Poly(dA) (Amersham, Piscataway, NJ); a 461 bp fragment of the kanamycin-resistant gene, amplified from the pET24b+ plasmid (Novagen, Madison, WI); PRRS virus strain VR-2332 (PRU87392) ORFs 2, 3, 4, 5, 6 and 7; pCMVSport 6 plasmid (Invitrogen, Carlsbad, CA); and pGEM-T plasmid (Promega, Madison WI). The mean intensity of the negative controls for both Cy3 and Cy5 was calculated for each slide, and spots with a mean intensity less than the mean intensity + 2SD were excluded from further analysis. After normalization, GeneSpring averaged replicate spots for each clone across hybridizations. GeneSpring used the Cross Gene Error Model, which was based on replicate values. A Student’s t-test with the Benjamini and Hochberg false discovery rate multiple testing correction (MTC) verified the difference between the natural log of the normalized gene expression ratio (JPP:j-MLN) and a ratio of 1.0. p-values less than 0.05 were considered significant. Series_references: 1.Fukushima K, Ogawa H, Takahashi K, Naito H, Funayama Y, Kitayama T, Yonezawa H, and Sasaki I. Non-pathogenic bacteria modulate colonic epithelial gene expression in germ-free mice. Scand J Gastroenterol 38: 626-634, 2003. 2. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, and Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291: 881-884, 2001.

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Supervised Lowess normalization of comparative genome hybridization data: application to lactococcal strain comparisons.

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Supervised Lowess normalization of comparative genome hybridization data--application to lactococcal strain comparisons.

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