A head and neck cancer tumor response-specific gene signature for cisplatin, 5-fluorouracil induction chemotherapy fails with added taxanes.

BACKGROUND: It is a major clinical challenge to predict which patients, with advanced stage head and neck squamous cell carcinoma, will not exhibit a reduction in tumor size following induction chemotherapy in order to avoid toxic effects of ineffective chemotherapy and delays for instituting other therapeutic options. Further, it is of interest to know to what extent a gene signature, which identifies patients with tumors that will not respond to a particular induction chemotherapy, is applicable when additional chemotherapeutic agents are added to the regimen. METHODOLOGY/PRINCIPAL FINDINGS: To identify genes that predict tumor resistance to induction with cisplatin/5-fluorouracil (PF) or PF and a taxane, we analyzed patient tumor biopsies with whole genome microarrays and quantitative reverse transcriptase-PCR (TLDA) cards. A leave one out cross-validation procedure allowed evaluation of the prediction tool. A ten-gene microarray signature correctly classified 12/13 responders and 7/10 non-responders to PF (92% specificity, 82.6% accuracy). TLDA analysis (using the same classifier) of the patients correctly classified 12/12 responders and 8/10 non-responders (100% specificity, 90.9% accuracy). Further, TLDA analysis correctly predicted the response of 5 new patients and, overall, 12/12 responders and 13/15 non-responders (100% specificity, 92.6% accuracy). The protein products of the genes constituting the signature physically associate with 27 other proteins, involved in regulating gene expression, constituting an interaction network. In contrast, TLDA-based prediction (with the same gene signature) of responses to induction with PF and either of two taxanes was poor (0% specificity, 25% accuracy and 33.3% specificity, 25% accuracy). CONCLUSIONS/SIGNIFICANCE: Successful transfer of the microarray-based gene signature to an independent, PCR-based technology suggests that TLDA-based signatures could be a useful hospital-based technology for determining therapeutic options. Although highly specific for tumor responses to PF induction, the gene signature is unsuccessful when taxanes are added. The results illustrate the subtlety in developing "personalized medicine".

The aryl hydrocarbon receptor system.

The aryl hydrocarbon receptor (AhR) recognizes a large number of xenobiotics, such as polyaromatic hydrocarbons (PAHs) and dioxins, and it activates several metabolic and detoxification pathways. Recent evidence suggests that this receptor also has important endogenous functions subsequent to activation by natural dietary compounds and/or endogenous metabolites. This receptor, thus, has physiological functions that extend beyond specific instances of detoxification. Understanding the roles played by this receptor might be enhanced by a systems biology approach. Indeed, the AhR "ligandome" is very complex and the different classes of ligands involved could induce widely diverse effects. The protein "interactome" of the AhR comprises several tens of proteins and it is altered by the binding of ligands to the receptor. Furthermore, large-scale studies have shown cell and tissue-specific patterns of regulated gene expression which may depend upon the type of ligand, although these aspects need further substantiation. Finally, the AhR biological effects are extensive and include detoxification, cellular proliferation and migration, immune regulation and neuronal effects. A holistic approach should provide a better understanding of the biology of this receptor in addition to providing new avenues for the identification of specific toxicity mechanisms.

Molecular and functional analysis of two new MTTP gene mutations in an atypical case of abetalipoproteinemia.

Abetalipoproteinemia (ABL) is an inherited disease characterized by the defective assembly and secretion of apolipoprotein B-containing lipoproteins caused by mutations in the microsomal triglyceride transfer protein large subunit (MTP) gene (MTTP). We report here a female patient with an unusual clinical and biochemical ABL phenotype. She presented with severe liver injury, low levels of LDL-cholesterol, and subnormal levels of vitamin E, but only mild fat malabsorption and no retinitis pigmentosa or acanthocytosis. Our objective was to search for MTTP mutations and to determine the relationship between the genotype and this particular phenotype. The subject exhibited compound heterozygosity for two novel MTTP mutations: one missense mutation (p.Leu435His) and an intronic deletion (c.619-5_619-2del). COS-1 cells expressing the missense mutant protein exhibited negligible levels of MTP activity. In contrast, the minigene splicing reporter assay showed an incomplete splicing defect of the intronic deletion, with 26% of the normal splicing being maintained in the transfected HeLa cells. The small amount of MTP activity resulting from the residual normal splicing in the patient explains the atypical phenotype observed. Our investigation provides an example of a functional analysis of unclassified variations, which is an absolute necessity for the molecular diagnosis of atypical ABL cases.

Anderson's disease/chylomicron retention disease in a Japanese patient with uniparental disomy 7 and a normal SAR1B gene protein coding sequence.

BACKGROUND: Anderson's Disease (AD)/Chylomicron Retention Disease (CMRD) is a rare hereditary hypocholesterolemic disorder characterized by a malabsorption syndrome with steatorrhea, failure to thrive and the absence of chylomicrons and apolipoprotein B48 post-prandially. All patients studied to date exhibit a mutation in the SAR1B gene, which codes for an essential component of the vesicular coat protein complex II (COPII) necessary for endoplasmic reticulum to Golgi transport. We describe here a patient with AD/CMRD, a normal SAR1B gene protein coding sequence and maternal uniparental disomy of chromosome 7 (matUPD7). METHODS AND RESULTS: The patient, one of two siblings of a Japanese family, had diarrhea and steatorrhea beginning at five months of age. There was a white duodenal mucosa upon endoscopy. Light and electron microscopy showed that the intestinal villi were normal but that they had lipid laden enterocytes containing accumulations of lipid droplets in the cytoplasm and lipoprotein-size particles in membrane bound structures. Although there were decreased amounts in plasma of total- and low-density lipoprotein cholesterol, apolipoproteins AI and B and vitamin E levels, the triglycerides were normal, typical of AD/CMRD. The presence of low density lipoproteins and apolipoprotein B in the plasma, although in decreased amounts, ruled out abetalipoproteinemia. The parents were asymptomatic with normal plasma cholesterol levels suggesting a recessive disorder and ruling out familial hypobetalipoproteinemia. Sequencing of genomic DNA showed that the 8 exons of the SAR1B gene were normal. Whole genome SNP analysis and karyotyping revealed matUPD7 with a normal karyotype. In contrast to other cases of AD/CMRD which have shown catch-up growth following vitamin supplementation and a fat restricted diet, our patient exhibits continued growth delay and other aspects of the matUPD7 and Silver-Russell Syndrome phenotypes. CONCLUSIONS: This patient with AD/CMRD has a normal SAR1B gene protein coding sequence which suggests that factors other than the SAR1B protein may be crucial for chylomicron secretion. Further, this patient exhibits matUPD7 with regions of homozygosity which might be useful for elucidating the molecular basis of the defect(s) in this individual. The results provide novel insights into the relation between phenotype and genotype in these diseases and for the mechanisms of secretion in the intestine.

Functional study of genes essential for autogamy and nuclear reorganization in Paramecium.

Like all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germ line micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy, and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by noncoding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 upregulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old and then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis.

Molecular analysis and intestinal expression of SAR1 genes and proteins in Anderson's disease (Chylomicron retention disease).

BACKGROUND: Anderson's disease (AD) or chylomicron retention disease (CMRD) is a very rare hereditary lipid malabsorption syndrome. In order to discover novel mutations in the SAR1B gene and to evaluate the expression, as compared to healthy subjects, of the Sar1 gene and protein paralogues in the intestine, we investigated three previously undescribed individuals with the disease. METHODS: The SAR1B, SAR1A and PCSK9 genes were sequenced. The expression of the SAR1B and SAR1A genes in intestinal biopsies of both normal individuals and patients was measured by RTqPCR. Immunohistochemistry using antibodies to recombinant Sar1 protein was used to evaluate the expression and localization of the Sar1 paralogues in the duodenal biopsies. RESULTS: Two patients had a novel SAR1B mutation (p.Asp48ThrfsX17). The third patient, who had a previously described SAR1B mutation (p.Leu28ArgfsX7), also had a p.Leu21dup variant of the PCSK9 gene. The expression of the SAR1B gene in duodenal biopsies from an AD/CMRD patient was significantly decreased whereas the expression of the SAR1A gene was significantly increased, as compared to healthy individuals. The Sar1 proteins were present in decreased amounts in enterocytes in duodenal biopsies from the patients as compared to those from healthy subjects. CONCLUSIONS: Although the proteins encoded by the SAR1A and SAR1B genes are 90% identical, the increased expression of the SAR1A gene in AD/CMRD does not appear to compensate for the lack of the SAR1B protein. The PCSK9 variant, although reported to be associated with low levels of cholesterol, does not appear to exert any additional effect in this patient. The results provide further insight into the tissue-specific nature of AD/CMRD.

SMARCA2 and other genome-wide supported schizophrenia-associated genes: regulation by REST/NRSF, network organization and primate-specific evolution.

The SMARCA2 gene, which encodes BRM in the SWI/SNF chromatin-remodeling complex, was recently identified as being associated with schizophrenia (SZ) in a genome-wide approach. Polymorphisms in SMARCA2, associated with the disease, produce changes in the expression of the gene and/or in the encoded amino acid sequence. We show here that an SWI/SNF-centered network including the Smarca2 gene is modified by the down-regulation of REST/NRSF in a mouse neuronal cell line. REST/NRSF down-regulation also modifies the levels of Smarce1, Smarcd3 and SWI/SNF interactors (Hdac1, RcoR1 and Mecp2). Smarca2 down-regulation generates an abnormal dendritic spine morphology that is an intermediate phenotype of SZ. We further found that 8 (CSF2RA, HIST1H2BJ, NOTCH4, NRGN, SHOX, SMARCA2, TCF4 and ZNF804A) out of 10 genome-wide supported SZ-associated genes are part of an interacting network (including SMARCA2), 5 members of which encode transcription regulators. The expression of 3 (TCF4, SMARCA2 and CSF2RA) of the 10 genome-wide supported SZ-associated genes is modified when the REST/NRSF-SWI/SNF chromatin-remodeling complex is experimentally manipulated in mouse cell lines and in transgenic mouse models. The REST/NRSF-SWI/SNF deregulation also results in the differential expression of genes that are clustered in chromosomes suggesting the induction of genome-wide epigenetic changes. Finally, we found that SMARCA2 interactors and the genome-wide supported SZ-associated genes are considerably enriched in genes displaying positive selection in primates and in the human lineage which suggests the occurrence of novel protein interactions in primates. Altogether, these data identify the SWI/SNF chromatin-remodeling complex as a key component of the genetic architecture of SZ.

Differentiation of symbiotic cells and endosymbionts in Medicago truncatula nodulation are coupled to two transcriptome-switches.

The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix(-) nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this "nodule-specific transcriptome" were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches.

Characterisation and correction of signal fluctuations in successive acquisitions of microarray images.

BACKGROUND: There are many sources of variation in dual labelled microarray experiments, including data acquisition and image processing. The final interpretation of experiments strongly relies on the accuracy of the measurement of the signal intensity. For low intensity spots in particular, accurately estimating gene expression variations remains a challenge as signal measurement is, in this case, highly subject to fluctuations. RESULTS: To evaluate the fluctuations in the fluorescence intensities of spots, we used series of successive scans, at the same settings, of whole genome arrays. We measured the decrease in fluorescence and we evaluated the influence of different parameters (PMT gain, resolution and chemistry of the slide) on the signal variability, at the level of the array as a whole and by intensity interval. Moreover, we assessed the effect of averaging scans on the fluctuations. We found that the extent of photo-bleaching was low and we established that 1) the fluorescence fluctuation is linked to the resolution e.g. it depends on the number of pixels in the spot 2) the fluorescence fluctuation increases as the scanner voltage increases and, moreover, is higher for the red as opposed to the green fluorescence which can introduce bias in the analysis 3) the signal variability is linked to the intensity level, it is higher for low intensities 4) the heterogeneity of the spots and the variability of the signal and the intensity ratios decrease when two or three scans are averaged. CONCLUSION: Protocols consisting of two scans, one at low and one at high PMT gains, or multiple scans (ten scans) can introduce bias or be difficult to implement. We found that averaging two, or at most three, acquisitions of microarrays scanned at moderate photomultiplier settings (PMT gain) is sufficient to significantly improve the accuracy (quality) of the data and particularly those for spots having low intensities and we propose this as a general approach. For averaging and precise image alignment at sub-pixel levels we have made a program freely available on our web-site http://bioinfome.cgm.cnrs-gif.fr to facilitate implementation of this approach.

DYRK1A interacts with the REST/NRSF-SWI/SNF chromatin remodelling complex to deregulate gene clusters involved in the neuronal phenotypic traits of Down syndrome.

The molecular mechanisms that lead to the cognitive defects characteristic of Down syndrome (DS), the most frequent cause of mental retardation, have remained elusive. Here we use a transgenic DS mouse model (152F7 line) to show that DYRK1A gene dosage imbalance deregulates chromosomal clusters of genes located near neuron-restrictive silencer factor (REST/NRSF) binding sites. We found that Dyrk1a binds the SWI/SNF complex known to interact with REST/NRSF. The mutation of a REST/NRSF binding site in the promoter of the REST/NRSF target gene L1cam modifies the transcriptional effect of Dyrk1a-dosage imbalance on L1cam. Dyrk1a dosage imbalance perturbs Rest/Nrsf levels with decreased Rest/Nrsf expression in embryonic neurons and increased expression in adult neurons. Using transcriptome analysis of embryonic brain subregions of transgenic 152F7 mouse line, we identified a coordinated deregulation of multiple genes that are responsible for dendritic growth impairment present in DS. Similarly, Dyrk1a overexpression in primary mouse cortical neurons induced severe reduction of the dendritic growth and dendritic complexity. We propose that DYRK1A overexpression-related neuronal gene deregulation via disturbance of REST/NRSF levels, and the REST/NRSF-SWI/SNF chromatin remodelling complex, significantly contributes to the neural phenotypic changes that characterize DS.

The Sterolgene v0 cDNA microarray: a systemic approach to studies of cholesterol homeostasis and drug metabolism.

BACKGROUND: Cholesterol homeostasis and xenobiotic metabolism are complex biological processes, which are difficult to study with traditional methods. Deciphering complex regulation and response of these two processes to different factors is crucial also for understanding of disease development. Systems biology tools as are microarrays can importantly contribute to this knowledge and can also discover novel interactions between the two processes. RESULTS: We have developed a low density Sterolgene v0 cDNA microarray dedicated to studies of cholesterol homeostasis and drug metabolism in the mouse. To illustrate its performance, we have analyzed mouse liver samples from studies focused on regulation of cholesterol homeostasis and drug metabolism by diet, drugs and inflammation. We observed down-regulation of cholesterol biosynthesis during fasting and high-cholesterol diet and subsequent up-regulation by inflammation. Drug metabolism was down-regulated by fasting and inflammation, but up-regulated by phenobarbital treatment and high-cholesterol diet. Additionally, the performance of the Sterolgene v0 was compared to the two commercial high density microarray platforms: the Agilent cDNA (G4104A) and the Affymetrix MOE430A GeneChip. We hybridized identical RNA samples to the commercial microarrays and showed that the performance of Sterolgene is comparable to commercial arrays in terms of detection of changes in cholesterol homeostasis and drug metabolism. CONCLUSION: Using the Sterolgene v0 microarray we were able to detect important changes in cholesterol homeostasis and drug metabolism caused by diet, drugs and inflammation. Together with its next generations the Sterolgene microarrays represent original and dedicated tools enabling focused and cost effective studies of cholesterol homeostasis and drug metabolism. These microarrays have the potential of being further developed into screening or diagnostic tools.

Expression ratio evaluation in two-colour microarray experiments is significantly improved by correcting image misalignment.

MOTIVATION: Two-colour microarrays are widely used to perform transcriptome analysis. In most cases, it appears that the 'red' and 'green' images resulting from the scan of a microarray slide are slightly shifted one with respect to the other. To increase the robustness of the measurement of the fluorescent emission intensities, multiple acquisitions with the same or different PMT gains can be used. In these cases, a systematic correction of image shift is required. RESULTS: To accurately detect this shift, we first developed an approach using cross-correlation. Second, we evaluated the most appropriate interpolation method to be used to derive the registered image. Then, we quantified the effects of image shifts on spot quality, using two different quality estimators. Finally, we measured the benefits associated with a systematic image registration. In this study, we demonstrate that registering the two images prior to data extraction provides a more reliable estimate of the two colours' ratio and thus increases the accuracy of measurements of variations in gene expression. AVAILABILITY: http://bioinfome.cgm.cnrs-gif.fr/.

MAnGO: an interactive R-based tool for two-colour microarray analysis.

UNLABELLED: MAnGO (Microarray Analysis at the Gif/Orsay platform) is an interactive R-based tool for the analysis of two-colour microarray experiments. It is a compilation of various methods, which allows the user (1) to control data quality by detecting biases with a large number of visual representations, (2) to pre-process data (filtering and normalization) and (3) to carry out differential analyses. MAnGO is not only a 'turn-key' tool, oriented towards biologists but also a flexible and adaptable R script oriented towards bioinformaticians. AVAILABILITY: http://bioinfome.cgm.cnrs-gif.fr/.

Genomewide and biochemical analyses of DNA-binding activity of Cdc6/Orc1 and Mcm proteins in Pyrococcus sp.

The origin of DNA replication (oriC) of the hyperthermophilic archaeon Pyrococcus abyssi contains multiple ORB and mini-ORB repeats that show sequence similarities to other archaeal ORB (origin recognition box). We report here that the binding of Cdc6/Orc1 to a 5 kb region containing oriC in vivo was highly specific both in exponential and stationary phases, by means of chromatin immunoprecipitation coupled with hybridization on a whole genome microarray (ChIP-chip). The oriC region is practically the sole binding site for the Cdc6/Orc1, thereby distinguishing oriC in the 1.8 M bp genome. We found that the 5 kb region contains a previously unnoticed cluster of ORB and mini-ORB repeats in the gene encoding the small subunit (dp1) for DNA polymerase II (PolD). ChIP and the gel retardation analyses further revealed that Cdc6/Orc1 specifically binds both of the ORB clusters in oriC and dp1. The organization of the ORB clusters in the dp1 and oriC is conserved during evolution in the order Thermococcales, suggesting a role in the initiation of DNA replication. Our ChIP-chip analysis also revealed that Mcm alters the binding specificity to the oriC region according to the growth phase, consistent with its role as a licensing factor.

The structure of human apolipoprotein E2, E3 and E4 in solution 1. Tertiary and quaternary structure.

Three recombinant apoE isoforms fused with an amino-terminal extension of 43 amino acids were produced in a heterologous expression system in E. coli. Their state of association in aqueous phase was analyzed by size-exclusion liquid chromatography, sedimentation velocity and sedimentation equilibrium experiments. By liquid chromatography, all three isoforms consisted of three major species with Stokes radii of 4.0, 5.0 and 6.6 nm. Sedimentation velocity confirmed the presence of monomers, dimers and tetramers as major species of each isoform. The association schemes established by sedimentation equilibrium experiments corresponded to monomer-dimer-tetramer-octamer for apoE2, monomer-dimer-tetramer for apoE3 and monomer-dimer-tetramer-octamer for apoE4. Each of the three isoforms exhibits a distinct self-association pattern. The apolipoprotein multi-domain structure was mapped by limited proteolysis with trypsin, chymotrypsin, elastase, subtilisin and Staphylococcus aureus V8 protease. All five enzymes produced stable intermediates during the degradation of the three apoE isoforms, as described for plasma apoE3. The recombinant apoE isoforms, thus, consist of N- and C-terminal domains. The presence of the fusion peptide did not appear to alter the apolipoprotein tertiary organization. However, a 30 kDa amino-terminal fragment appeared during the degradation of the recombinant apoE isoforms resulting from cleavage in the 273-278 region. This region, not accessible in plasma apoE3, results from a different conformation of the C-terminal domain in the recombinant isoforms. A specific pattern for the apoE4 C-terminal domain was observed during the proteolysis. The region 230-260 in apoE4, in contrast to that of apoE3 and apoE2, was not accessible to proteases, probably due to the existence of a longer helix in this region of apoE4 stabilized by an interdomain interaction.

The structure of human apolipoprotein E2, E3 and E4 in solution. 2. Multidomain organization correlates with the stability of apoE structure.

The stabilities toward thermal and chemical denaturation of three recombinant isoforms of human apolipoprotein E (r-apoE2, r-apoE3 and r-apoE4), human plasma apoE3, the recombinant amino-terminal (NT) and the carboxyl-terminal (CT) domains of plasma apoE3 at pH 7 were studied using near and far ultraviolet circular dichroism (UV CD), fluorescence and size-exclusion chromatography. By far UV CD, thermal unfolding was irreversible for the intact apoE isoforms and consisted of a single transition. The r-apoE3 was found to be less stable as compared to the plasma protein and the stability of recombinant isoforms was r-apoE4

Optimizing spotting solutions for increased reproducibility of cDNA microarrays.

The ability to extract meaningful information from transcriptome technologies such as cDNA microarrays relies on the precision, sensitivity and reproducibility of the measured values for a given gene across multiple samples. Given the lack of a 'gold standard' for the production of microarrays using current technologies, there is a high degree of variation in the quality of data derived from microarray experiments. Poor reproducibility not only adds to the cost of a given study but also leads to data sets that are difficult to interpret. For glass slide DNA microarrays, much of this variation is introduced systematically, during the spotting, or deposition, of the DNA onto the slide surface. In order to reduce this type of systematic variation we tested spotting solutions containing different detergent additives in the presence of one of two different denaturants and determined their effect on spot quality. We show that spotting cDNA in a solution consisting of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in the presence of formamide or dimethyl sulfoxide yields spots of superior quality in terms of morphology, size homogeneity and signal reproducibility, as well as overall intensity, when used with popular, commercially available slides.

Computational methods for sequence mapping of large combinatorial libraries and deduced sequence signatures.

Here we describe a computational approach for the high-throughput sequence mapping of combinatorial libraries obtained by DNA shuffling. Original algorithms and their software implementation were developed for the automated and reliable analysis of hybridization data of differentially labeled oligonucleotide probes with PCR products spotted on DNA microarrays. This novel approach allows a context-dependent sequence attribution tolerant to fluctuations in experimental conditions and is well adapted to hybridization signals of variable qualities resulting from high-throughput PCR amplification from colonies. In addition, the analysis permits the calculation of sequence signatures that are characteristic of combinatorial library structure, defects, and diversity. The approach is of interest for the characterization and the equalization (library reduction to nonredundant structures) of combinatorial libraries involved in directed evolution and could be extrapolated to high-throughput polymorphism analysis.