Novel genotyping algorithms for rare variants significantly improve the accuracy of Applied Biosystems™ Axiom™ array genotyping calls: Retrospective evaluation of UK Biobank array data.

The UK Biobank genotyped about 500k participants using Applied Biosystems Axiom microarrays. Participants were subsequently sequenced by the UK Biobank Exome Sequencing Consortium. Axiom genotyping was highly accurate in comparison to sequencing results, for almost 100,000 variants both directly genotyped on the UK Biobank Axiom array and via whole exome sequencing. However, in a study using the exome sequencing results of the first 50k individuals as reference (truth), it was observed that the positive predictive value (PPV) decreased along with the number of heterozygous array calls per variant. We developed a novel addition to the genotyping algorithm, Rare Heterozygous Adjusted (RHA), to significantly improve PPV in variants with minor allele frequency below 0.01%. The improvement in PPV was roughly equal when comparing to the exome sequencing of 50k individuals, or to the more recent ~200k individuals. Sensitivity was higher in the 200k data. The improved calling algorithm, along with enhanced quality control of array probesets, significantly improved the positive predictive value and the sensitivity of array data, making it suitable for the detection of ultra-rare variants.

Development and validation of a universal blood donor genotyping platform: a multinational prospective study.

Each year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non-self-antigens is an unavoidable adverse side effect of transfusion. We describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.

Genotyping Informatics and Quality Control for 100,000 Subjects in the Genetic Epidemiology Research on Adult Health and Aging (GERA) Cohort.

The Kaiser Permanente (KP) Research Program on Genes, Environment and Health (RPGEH), in collaboration with the University of California-San Francisco, undertook genome-wide genotyping of >100,000 subjects that constitute the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort. The project, which generated >70 billion genotypes, represents the first large-scale use of the Affymetrix Axiom Genotyping Solution. Because genotyping took place over a short 14-month period, creating a near-real-time analysis pipeline for experimental assay quality control and final optimized analyses was critical. Because of the multi-ethnic nature of the cohort, four different ethnic-specific arrays were employed to enhance genome-wide coverage. All assays were performed on DNA extracted from saliva samples. To improve sample call rates and significantly increase genotype concordance, we partitioned the cohort into disjoint packages of plates with similar assay contexts. Using strict QC criteria, the overall genotyping success rate was 103,067 of 109,837 samples assayed (93.8%), with a range of 92.1-95.4% for the four different arrays. Similarly, the SNP genotyping success rate ranged from 98.1 to 99.4% across the four arrays, the variation depending mostly on how many SNPs were included as single copy vs. double copy on a particular array. The high quality and large scale of genotype data created on this cohort, in conjunction with comprehensive longitudinal data from the KP electronic health records of participants, will enable a broad range of highly powered genome-wide association studies on a diversity of traits and conditions.

MicroRNA polymorphisms and risk of colorectal cancer.

BACKGROUND: miRNAs act as post-transcriptional regulators of gene expression. Genetic variation in miRNA-encoding sequences or their corresponding binding sites may affect the fidelity of the miRNA-mRNA interaction and subsequently alter the risk of cancer development. METHODS: This study expanded the search for miRNA-related polymorphisms contributing to the etiology of colorectal cancer across the genome using a novel platform, the Axiom miRNA Target Site Genotyping Array (237,858 markers). After quality control, the study included 596 cases and 429 controls from the Molecular Epidemiology of Colorectal Cancer study, a population-based case-control study of colorectal cancer in northern Israel. The association between each marker and colorectal cancer status was examined assuming a log-additive genetic model using logistic regression adjusted for sex, age, and two principal components. RESULTS: Twenty-three markers had P values less than 5.0E-04, and the most statistically significant association involved rs2985 (chr6:34845648; intronic of UHRF1BP1; OR = 0.66; P = 3.7E-05). Furthermore, this study replicated a previously published risk locus, rs1051690, in the 3'-untranslated region of the insulin receptor gene INSR (OR = 1.38; P = 0.03), with strong evidence of differences in INSR gene expression by genotype. CONCLUSIONS: This study is the first to examine associations between genetic variation in miRNA target sites and colorectal cancer using a genome-wide approach. Functional studies to identify allele-specific effects on miRNA binding are needed to confirm the regulatory capacity of genetic variation to influence risk of colorectal cancer. IMPACT: This study demonstrates the potential for an miRNA-targeted genome-wide association study to identify candidate susceptibility loci and prioritize them for functional characterization.

Genome-wide maps of circulating miRNA biomarkers for ulcerative colitis.

Inflammatory Bowel Disease--comprised of Crohn's Disease and Ulcerative Colitis (UC)--is a complex, multi-factorial inflammatory disorder of the gastrointestinal tract. In this study we have explored the utility of naturally occurring circulating miRNAs as potential blood-based biomarkers for non-invasive prediction of UC incidences. Whole genome maps of circulating miRNAs in micro-vesicles, Peripheral Blood Mononuclear Cells and platelets have been constructed from a cohort of 20 UC patients and 20 normal individuals. Through Significance Analysis of Microarrays, a signature of 31 differentially expressed platelet-derived miRNAs has been identified and biomarker performance estimated through a non-probabilistic binary linear classification using Support Vector Machines. Through this approach, classifier measurements reveal a predictive score of 92.8% accuracy, 96.2% specificity and 89.5% sensitivity in distinguishing UC patients from normal individuals. Additionally, the platelet-derived biomarker signature can be validated at 88% accuracy through qPCR assays, and a majority of the miRNAs in this panel can be demonstrated to sub-stratify into 4 highly correlated intensity based clusters. Analysis of predicted targets of these biomarkers reveal an enrichment of pathways associated with cytoskeleton assembly, transport, membrane permeability and regulation of transcription factors engaged in a variety of regulatory cascades that are consistent with a cell-mediated immune response model of intestinal inflammation. Interestingly, comparison of the miRNA biomarker panel and genetic loci implicated in IBD through genome-wide association studies identifies a physical linkage between hsa-miR-941 and a UC susceptibility loci located on Chr 20. Taken together, analysis of these expression maps outlines a promising catalog of novel platelet-derived miRNA biomarkers of clinical utility and provides insight into the potential biological function of these candidates in disease pathogenesis.

Design and coverage of high throughput genotyping arrays optimized for individuals of East Asian, African American, and Latino race/ethnicity using imputation and a novel hybrid SNP selection algorithm.

Four custom Axiom genotyping arrays were designed for a genome-wide association (GWA) study of 100,000 participants from the Kaiser Permanente Research Program on Genes, Environment and Health. The array optimized for individuals of European race/ethnicity was previously described. Here we detail the development of three additional microarrays optimized for individuals of East Asian, African American, and Latino race/ethnicity. For these arrays, we decreased redundancy of high-performing SNPs to increase SNP capacity. The East Asian array was designed using greedy pairwise SNP selection. However, removing SNPs from the target set based on imputation coverage is more efficient than pairwise tagging. Therefore, we developed a novel hybrid SNP selection method for the African American and Latino arrays utilizing rounds of greedy pairwise SNP selection, followed by removal from the target set of SNPs covered by imputation. The arrays provide excellent genome-wide coverage and are valuable additions for large-scale GWA studies.

Impact of cellular miRNAs on circulating miRNA biomarker signatures.

Effective diagnosis and surveillance of complex multi-factorial disorders such as cancer can be improved by screening of easily accessible biomarkers. Highly stable cell free Circulating Nucleic Acids (CNA) present as both RNA and DNA species have been discovered in the blood and plasma of humans. Correlations between tumor-associated genomic/epigenetic/transcriptional changes and alterations in CNA levels are strong predictors of the utility of this biomarker class as promising clinical indicators. Towards this goal microRNAs (miRNAs) representing a class of naturally occurring small non-coding RNAs of 19-25 nt in length have emerged as an important set of markers that can associate their specific expression profiles with cancer development. In this study we investigate some of the pre-analytic considerations for isolating plasma fractions for the study of miRNA biomarkers. We find that measurement of circulating miRNA levels are frequently confounded by varying levels of cellular miRNAs of different hematopoietic origins. In order to assess the relative proportions of this cell-derived class, we have fractionated whole blood into plasma and its ensuing sub-fractions. Cellular miRNA signatures in cohorts of normal individuals are catalogued and the abundance and gender specific expression of bona fide circulating markers explored after calibrating the signal for this interfering class. A map of differentially expressed profiles is presented and the intrinsic variability of circulating miRNA species investigated in subsets of healthy males and females.

Next generation genome-wide association tool: design and coverage of a high-throughput European-optimized SNP array.

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.

Erratum to "Prediction of non-genotoxic carcinogenesis in rats using changes in gene expression following acute dosing".

Carcinogenicity of chemicals can currently only be evaluated in 2-year rodent bioassays. Therefore, the development of early biomarkers for carcinogenesis would result in substantial savings in time and expense. The current study investigates whether early changes in gene expression may be developed as markers for cancer. Animals were treated for 1 or 5 days with either non-genotoxic carcinogens or non-carcinogens and gene expression was analyzed by quantitative PCR (qPCR).We tested two gene signatures previously reported to detect non-genotoxic carcinogens. Using one gene signature it was confirmed that 3/3 nongenotoxic carcinogens and 2/2 non-carcinogens are correctly identified with data from 1 or 5 days of dosing. In contrast an alternative signature correctly identified 0/3 and 2/3 nongenotoxic carcinogens at 1 and 5 days of treatment, respectively and 2/2 non-carcinogens at both time-points. Additionally, we evaluated a novel panel of putative biomarker genes, from the literature, many of which have roles in cell growth and division, including myc, cdc2 and mcm6. These genes were significantly induced by non-genotoxic carcinogens and not by non-carcinogens. Using the average fold-induction across this panel, 2/3 non-genotoxic carcinogens were detected on both day 1 and day 5. These data support the idea that acute changes in gene expression may provide biomarkers for non-genotoxic carcinogenesis but also highlight interesting differences in the sensitivities of distinct gene signatures.

The liver pharmacological and xenobiotic gene response repertoire.

We have used a supervised classification approach to systematically mine a large microarray database derived from livers of compound-treated rats. Thirty-four distinct signatures (classifiers) for pharmacological and toxicological end points can be identified. Just 200 genes are sufficient to classify these end points. Signatures were enriched in xenobiotic and immune response genes and contain un-annotated genes, indicating that not all key genes in the liver xenobiotic responses have been characterized. Many signatures with equal classification capabilities but with no gene in common can be derived for the same phenotypic end point. The analysis of the union of all genes present in these signatures can reveal the underlying biology of that end point as illustrated here using liver fibrosis signatures. Our approach using the whole genome and a diverse set of compounds allows a comprehensive view of most pharmacological and toxicological questions and is applicable to other situations such as disease and development.

Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat.

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints. The Carcinogenicity Working Group of the Predictive Safety Testing Consortium has concentrated on sharing data to test the predictivity of two published hepatic gene expression signatures, including the signature by Fielden et al. (2007, Toxicol. Sci. 99, 90-100) for predicting nongenotoxic hepatocarcinogens, and the signature by Nie et al. (2006, Mol. Carcinog. 45, 914-933) for predicting nongenotoxic carcinogens. Although not a rigorous prospective validation exercise, the consortium approach created an opportunity to perform a meta-analysis to evaluate microarray data from short-term rat studies on over 150 compounds. Despite significant differences in study designs and microarray platforms between laboratories, the signatures proved to be relatively robust and more accurate than expected by chance. The accuracy of the Fielden et al. signature was between 63 and 69%, whereas the accuracy of the Nie et al. signature was between 55 and 64%. As expected, the predictivity was reduced relative to internal validation estimates reported under identical test conditions. Although the signatures were not deemed suitable for use in regulatory decision making, they were deemed worthwhile in the early assessment of drugs to aid decision making in drug development. These results have prompted additional efforts to rederive and evaluate a QPCR-based signature using these samples. When combined with a standardized test procedure and prospective interlaboratory validation, the accuracy and potential utility in preclinical applications can be ascertained.

Radiogenomic analysis to identify imaging phenotypes associated with drug response gene expression programs in hepatocellular carcinoma.

PURPOSE: To determine whether conventional contrast-enhanced computed tomography (CT) could be used to identify imaging phenotypes associated with a doxorubicin drug response gene expression program in hepatocellular carcinoma (HCC) by using an integrated imaging-genomic approach. MATERIALS AND METHODS: Thirty HCCs were analyzed and scored individually across six predefined imaging phenotypes. Unsupervised and supervised bioinformatics analyses were performed to correlate the imaging scores with the corresponding tumor microarray data (each microarray contained gene expression measurements across approximately 18,000 genes) to identify relationships between the imaging traits and underlying tumor gene expression. Enrichment for a predefined doxorubicin-response gene expression program was then performed against the imaging phenotype-associated genes and enrichment determined. RESULTS: An imaging phenotype related to tumor margins on arterial phase images demonstrated significant correlation with the doxorubicin-response transcriptional program (P < .05, q < 0.1). It was also significantly associated with HCC venous invasion and tumor stage (P < .05, q < 0.1). Tumors with higher tumor margin scores were more strongly associated with the doxorubicin resistance transcriptional program and had a greater prevalence of venous invasion and worse stage. Tumors with lower tumor margin scores, however, demonstrated a converse relationship. CONCLUSIONS: It is possible to identify HCC imaging phenotypes at CT that correlate with a doxorubicin drug response gene expression program. Given the role of doxorubicin in regional therapies for HCC management, it is possible that such an approach could be used to guide HCC therapy on a tumor-by-tumor basis on the basis of underlying tumor gene expression patterns.

A gene expression biomarker provides early prediction and mechanistic assessment of hepatic tumor induction by nongenotoxic chemicals.

There are currently no accurate and well-validated short-term tests to identify nongenotoxic hepatic tumorigens, thus necessitating an expensive 2-year rodent bioassay before a risk assessment can begin. Using hepatic gene expression data from rats treated for 5 days with one of 100 structurally and mechanistically diverse nongenotoxic hepatocarcinogens and nonhepatocarcinogens, a novel multigenebiomarker (i.e., signature) was derived to predict the likelihood of nongenotoxic chemicals to induce liver tumors in longer term studies. Independent validation of the signature on 47 test chemicals indicates an assay sensitivity and specificity of 86% and 81%, respectively. Alternate short-term in vivo pathological and genomic biomarkers were evaluated in parallel for comparison, including liver weight, hepatocellular hypertrophy, hepatic necrosis, serum alanine aminotransferase activity, induction of cytochrome P450 genes, and repression of Tsc-22 or alpha2-macroglobulin messenger RNA. In contrast to these biomarkers, the gene expression-based signature was more accurate. Unlike existing tests, an understanding of potential modes of action for hepatic tumorigenicity can be derived by comparison of the signature profile of test chemicals to hepatic tumorigens of known mechanism, including regenerative proliferation, proliferation associated with xenobiotic receptor activation, peroxisome proliferation, and steroid hormone-mediated mechanisms. This signature is not only more accurate than current methods, but also facilitates the identification of mode of action to aid in the early assessment of human cancer risk.

Decoding global gene expression programs in liver cancer by noninvasive imaging.

Paralleling the diversity of genetic and protein activities, pathologic human tissues also exhibit diverse radiographic features. Here we show that dynamic imaging traits in non-invasive computed tomography (CT) systematically correlate with the global gene expression programs of primary human liver cancer. Combinations of twenty-eight imaging traits can reconstruct 78% of the global gene expression profiles, revealing cell proliferation, liver synthetic function, and patient prognosis. Thus, genomic activity of human liver cancers can be decoded by noninvasive imaging, thereby enabling noninvasive, serial and frequent molecular profiling for personalized medicine.

The Stanford Microarray Database: implementation of new analysis tools and open source release of software.

The Stanford Microarray Database (SMD; http://smd.stanford.edu/) is a research tool and archive that allows hundreds of researchers worldwide to store, annotate, analyze and share data generated by microarray technology. SMD supports most major microarray platforms, and is MIAME-supportive and can export or import MAGE-ML. The primary mission of SMD is to be a research tool that supports researchers from the point of data generation to data publication and dissemination, but it also provides unrestricted access to analysis tools and public data from 300 publications. In addition to supporting ongoing research, SMD makes its source code fully and freely available to others under an Open Source license, enabling other groups to create a local installation of SMD. In this article, we describe several data analysis tools implemented in SMD and we discuss features of our software release.

Clustering microarray data.

Even a simple, small-scale, microarray experiment generates thousands to millions of data points. Clearly, spreadsheets or plotting programs do not suffice for analysis of such large volumes of data, and comprehensive analysis requires systematic methods for selection and organization of data. This chapter focuses on the concepts and algorithms of hierarchical clustering and the most commonly employed methods of partitioning or organizing microarray data, and freely available software that implements these algorithms.

The Stanford Microarray Database: a user's guide.

The Stanford Microarray Database (SMD) is a DNA microarray research database that provides a large amount of data for public use. This chapter describes the use of the primary tools for searching, browsing, retrieving, and analyzing data available for SMD. With this introduction, researchers and students will be able to examine and analyze a large body of gene expression and other experiments. Additional tools for depositing, annotating, sharing, and analyzing data, available only to registered users, are also described. SMD is available for installation as a local database.

The Stanford Microarray Database accommodates additional microarray platforms and data formats.

The Stanford Microarray Database (SMD) (http://smd.stanford.edu) is a research tool for hundreds of Stanford researchers and their collaborators. In addition, SMD functions as a resource for the entire biological research community by providing unrestricted access to microarray data published by SMD users and by disseminating its source code. In addition to storing GenePix (Axon Instruments) and ScanAlyze output from spotted microarrays, SMD has recently added the ability to store, retrieve, display and analyze the complete raw data produced by several additional microarray platforms and image analysis software packages, so that we can also now accept data from Affymetrix GeneChips (MAS5/GCOS or dChip), Agilent Catalog or Custom arrays (using Agilent's Feature Extraction software) or data created by SpotReader (Niles Scientific). We have implemented software that allows us to accept MAGE-ML documents from array manufacturers and to submit MIAME-compliant data in MAGE-ML format directly to ArrayExpress and GEO, greatly increasing the ease with which data from SMD can be published adhering to accepted standards and also increasing the accessibility of published microarray data to the general public. We have introduced a new tool to facilitate data sharing among our users, so that datasets can be shared during, before or after the completion of data analysis. The latest version of the source code for the complete database package was released in November 2004 (http://smd.stanford.edu/download/), allowing researchers around the world to deploy their own installations of SMD.

GO::TermFinder--open source software for accessing Gene Ontology information and finding significantly enriched Gene Ontology terms associated with a list of genes.

SUMMARY: GO::TermFinder comprises a set of object-oriented Perl modules for accessing Gene Ontology (GO) information and evaluating and visualizing the collective annotation of a list of genes to GO terms. It can be used to draw conclusions from microarray and other biological data, calculating the statistical significance of each annotation. GO::TermFinder can be used on any system on which Perl can be run, either as a command line application, in single or batch mode, or as a web-based CGI script. AVAILABILITY: The full source code and documentation for GO::TermFinder are freely available from http://search.cpan.org/dist/GO-TermFinder/.

The Stanford Microarray Database: data access and quality assessment tools.

The Stanford Microarray Database (SMD; http://genome-www.stanford.edu/microarray/) serves as a microarray research database for Stanford investigators and their collaborators. In addition, SMD functions as a resource for the entire scientific community, by making freely available all of its source code and providing full public access to data published by SMD users, along with many tools to explore and analyze those data. SMD currently provides public access to data from 3500 microarrays, including data from 85 publications, and this total is increasing rapidly. In this article, we describe some of SMD's newer tools for accessing public data, assessing data quality and for data analysis.