CiteULike: jyuh's snp

Less is more, except when less is less: Studying joint effects.

C R Weinberg — 2008-07-25T14:43:13-00:00

Genomics (18 July 2008)

Most diseases are complex in that they are caused by the joint action of multiple factors, both genetic and environmental. Over the past few decades, the mathematical convenience of logistic regression has served to enshrine the multiplicative model, to the point where many epidemiologists believe that departure from additivity on a log scale implies that two factors interact in causing disease. Other terminology in epidemiology, where students are told that inequality of relative risks across levels of a second factor should be seen as "effect modification," reinforces an uncritical acceptance of multiplicative joint effect as the biologically meaningful no-interaction null. Our first task, when studying joint effects, is to understand the limitations of our definitions for "interaction," and recognize that what statisticians mean and what biologists might want to mean by interaction may not coincide. Joint effects are notoriously hard to identify and characterize, even when asking a simple and unsatisfying question, like whether two effects are log-additive. The rule of thumb for such efforts is that a factor-of-four sample size is needed, compared with that needed to demonstrate main effects of either genes or exposures. So strategies have been devised that focus on the most informative individuals, either through risk-based sampling for a cohort, or case-control sampling, extreme phenotype sampling, pooling, two-stage sampling, exposed-only, or case-only designs. These designs gain efficiency, but at a cost of flexibility in models for joint effects. A relatively new approach avoids population controls by genotyping case-parent triads. Because it requires parents, the method works best for diseases with onset early in life. With this design, the role of autosomal genetic variants is assessed by in effect treating the nontransmitted parental alleles as controls for affected offspring. Despite advantages for looking at genetic effects, the triad design faces limitations when examining joint effects of genetic and environmental factors. Because population-based controls are not included, main effects for exposures cannot be estimated, and consequently one only has access to inference related to a multiplicative null. We have proposed a hybrid approach that offers the best features of both case-parent and case-control designs. Through genotyping of parents of population-based controls and assuming Mendelian transmission, power is markedly enhanced. One can also estimate main effects for exposures and now flexibly assess models for joint effects.

Analysis of pooled DNA samples on high density arrays without prior knowledge of differential hybridization rates.

S Macgregor — 2008-07-25T10:01:34-00:00

Nucleic acids research, Vol. 34, No. 7. (2006)

Array based DNA pooling techniques facilitate genome-wide scale genotyping of large samples. We describe a structured analysis method for pooled data using internal replication information in large scale genotyping sets. The method takes advantage of information from single nucleotide polymorphisms (SNPs) typed in parallel on a high density array to construct a test statistic with desirable statistical properties. We utilize a general linear model to appropriately account for the structured multiple measurements available with array data. The method does not require the use of additional arrays for the estimation of unequal hybridization rates and hence scales readily to accommodate arrays with several hundred thousand SNPs. Tests for differences between cases and controls can be conducted with very few arrays. We demonstrate the method on 384 endometriosis cases and controls, typed using Affymetrix Genechip(c) HindIII 50 K arrays. For a subset of this data there were accurate measures of hybridization rates available. Assuming equal hybridization rates is shown to have a negligible effect upon the results. With a total of only six arrays, the method extracted one-third of the information (in terms of equivalent sample size) available with individual genotyping (requiring 768 arrays). With 20 arrays (10 for cases, 10 for controls), over half of the information could be extracted from this sample.

A model of higher accuracy for the individual haplotyping problem based on weighted SNP fragments and genotype with errors

Minzhu Xie — 2008-07-03T16:49:43-00:00

Bioinformatics, Vol. 24, No. 13. (1 July 2008), pp. i105-113.

Motivation: In genetic studies of complex diseases, haplotypes provide more information than genotypes. However, haplotyping is much more difficult than genotyping using biological techniques. Therefore effective computational techniques have been in demand. The individual haplotyping problem is the computational problem of inducing a pair of haplotypes from an individual's aligned SNP fragments. Based on various optimal criteria and including different extra information, many models for the problem have been proposed. Higher accuracy of the models has been an important issue in the study of haplotype reconstruction. Results: The current article proposes a highly accurate model for the single individual haplotyping problem based on weighted fragments and genotypes with errors. The model is proved to be NP-hard even with gapless fragments. Based on the characteristics of Single Nucleotide Polymorphism (SNP) fragments, a parameterized algorithm of time complexity O(nk22k2 + m log m + mk1) is developed, where m is the number of fragments, n is the number of SNP sites, k1 is the maximum number of SNP sites that a fragment covers (no more than n and usually smaller than 10) and k2 is the maximum number of the fragments covering a SNP site (usually no more than 19). Extensive experiments show that this model is more accurate in haplotype reconstruction than other models. Availability: The program of the parameterized algorithm can be obtained by sending an email to the corresponding author. Contact: jxwang@mail.csu.edu.cn 10.1093/bioinformatics/btn147

Genotype, haplotype and copy-number variation in worldwide human populations

Mattias Jakobsson — 2008-02-21T01:25:34-00:00

Nature, Vol. 451, No. 7181. (21 February 2008), pp. 998-1003.

Microarray technology and applications in the arena of genome-wide association.

SF Grant — 2008-07-16T12:21:13-00:00

Clinical chemistry, Vol. 54, No. 7. (July 2008), pp. 1116-1124.

BACKGROUND: There is a revolution occurring in single nucleotide polymorphism (SNP) genotyping technology, with high-throughput methods now allowing large numbers of SNPs (10(5)-10(6)) to be genotyped in large cohort studies. This has enabled large-scale genome-wide association (GWA) studies in complex diseases, such as diabetes, asthma, and inflammatory bowel disease, to be undertaken for the first time. Content: The GWA approach serves the critical need for a comprehensive and unbiased strategy to identify causal genes related to complex disease, and is rapidly replacing the more traditional candidate gene studies and microsatellite-based linkage mapping approaches that have dominated gene discovery attempts for common diseases. As a consequence of employing array-based technologies, over the last 3 years dramatic discoveries of key variants involved in multiple complex diseases and related traits have been reported in the top scientific literature and, most importantly, have been largely replicated by independent investigator groups. As a consequence, several novel genes have been identified, most notably in the metabolic, cardiovascular, autoimmune, and oncology disease areas, that are clearly rooted in the biology of these disorders. These discoveries have opened up new avenues for investigators to address novel molecular pathways that were not previously linked to or thought of in relation with these diseases. Summary: This review provides a synopsis of recent advances and what we may expect to still emerge from this field.

An optimal method of DNA silver staining in polyacrylamide gels.

YC Han — 2008-07-23T16:48:00-00:00

Electrophoresis, Vol. 29, No. 6. (March 2008), pp. 1355-1358.

DNA silver staining has widely been used to detect DNA fragments in polyacrylamide gels with high sensitivity. We developed an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure can be completed within 10 min instead of over 20 min with the conventional methods. The sensitivity is significantly improved by the silver-ion sensitizer (Eriochrome black T (EBT)) and the minimum of 0.11 and 1.75 ng of DNA amount can be detected in denaturing and nondenaturing polyacrylamide gel, respectively. Compared with the conventional silver staining methods, the improved optimal method can save time and display high sensitivity, color uniformity, and long storage time of the staining gels.

An optimal method of DNA silver staining in polyacrylamide gels.

YT Ji — 2008-07-23T16:46:31-00:00

Electrophoresis, Vol. 28, No. 8. (April 2007), pp. 1173-1175.

A silver staining technique has widely been used to detect DNA fragments with high sensitivity on polyacrylamide gels. The conventional procedure of the silver staining is tedious, which takes about 40-60 min and needs five or six kinds of chemicals and four kinds of solutions. Although our previous improved method reduced several steps, it still needed six kinds of chemicals. The objective of this study was to improve further the existing procedures and develop an optimal method for DNA silver staining on polyacrylamide gels. The novel procedure could be completed with only four chemicals and two solutions within 20 min. The steps of ethanol, acetic acid, and nitric acid precession before silver impregnation have been eliminated and the minimal AgNO3 dose has been used in this up-to-date method. The polyacrylamide gel of the DNA silver staining displayed a golden yellow and transparent background with high sensitivity. The minimum 0.44 and 3.5 ng of DNA amount could be detected in denaturing and nondenaturing polyacrylamide gel, respectively. This result indicated that our optimal method can save time and cost, and still keep a high sensitivity for DNA staining in polyacrylamide gels.

Single-strand conformation polymorphism and heteroduplex analysis for gel-based mutation detection.

AJ Nataraj — 2006-03-30T12:06:33-00:00

Electrophoresis, Vol. 20, No. 6. (June 1999), pp. 1177-1185.

Single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) are popular electrophoretic methods for the identification of sequences. The principle reasons for the popularity of these two methods are their technical simplicity and their relatively high sensitivity for the detection of mutations. Here we review the theory and practice of SSCP and HA, including the factors contributing to the sensitivity of mutation detection. For SSCP analysis, these factors include: choice of gel matrix, electrophoretic conditions, presence of neutral additives, fragment size, and G+C content For HA, the principle factors influencing sensitivity are the gel matrix and the identity of the base mismatch.

Combined SSCP/heteroduplex analysis in the screening for PAX6 mutations.

RA Axton — 2008-07-23T16:44:10-00:00

Molecular and cellular probes, Vol. 11, No. 4. (August 1997), pp. 287-292.

We demonstrate the use of combined SSCP and heteroduplex analysis in the detection of PAX6 mutations using non-radioactive silver staining. A panel of aniridia patients was screened by this approach and we show that a greater number of mutations was detected than would have been found by running each technique alone. Six previously unreported aniridia mutations in PAX6 are also described..

SNPper: retrieval and analysis of human SNPs.

A Riva — 2008-07-22T01:17:19-00:00

Bioinformatics (Oxford, England), Vol. 18, No. 12. (December 2002), pp. 1681-1685.

MOTIVATION: Single Nucleotide Polymorphisms (SNPs) are an increasingly important tool for the study of the human genome. SNPs can be used as markers to create high-density genetic maps, as causal candidates for diseases, or to reconstruct the history of our genome. SNP-based studies rely on the availability of large numbers of validated, high-frequency SNPs whose position on the chromosomes is known with precision. Although large collections of SNPs exist in public databases, researchers need tools to effectively retrieve and manipulate them. RESULTS: We describe the implementation and usage of SNPper, a web-based application to automate the tasks of extracting SNPs from public databases, analyzing them and exporting them in formats suitable for subsequent use. Our application is oriented toward the needs of candidate-gene, whole-genome and fine-mapping studies, and provides several flexible ways to present and export the data. The application has been publicly available for over a year, and has received positive user feedback and high usage levels.

Screening human genes for small alterations performing an enzymatic cleavage mismatched analysis (ECMA) protocol.

N Vogiatzakis — 2008-07-20T01:04:30-00:00

Molecular biotechnology, Vol. 37, No. 3. (November 2007), pp. 212-219.

Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry's disease (FD), steroid 21-hydroxylase deficiency (21-HD) and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases, where other techniques have failed.

PCR-SSCP: a method for the molecular analysis of genetic diseases.

KV Konstantinos — 2008-07-20T01:02:57-00:00

Molecular biotechnology, Vol. 38, No. 2. (February 2008), pp. 155-163.

Single strand conformation polymorphism (SSCP) is a reproducible, rapid and quite simple method for the detection of deletions/insertions/rearrangements in polymerase chain reaction amplified DNA. All the details for the use of PCR-SSCP are presented in the direction of genetic diseases (beta-thalassaemia, cystic fibrosis), optimum gel conditions, sensitivity and the latest modifications of the method, which are applied in most laboratories. This non-radioactive PCR-SSCP method can be reliably used to identify mutations in patients (beta-globin, CFTR), provided suitable controls are available. Moreover, it is widely used for mutation identification in carriers (beta-thalassaemia, cystic fibrosis), making it particularly useful in population screening.

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

Takanori Tsuji — 2008-07-20T00:53:17-00:00

ELECTROPHORESIS, Vol. 29, No. 7. (2008), pp. 1473-1483.

Efficient screening of unknown DNA variations is one of the substantive matters of molecular biology even today. Historically, SSCP and heteroduplex analysis (HA) are the most commonly used methods for detecting DNA variations everywhere in the world because of their simplicity. However, the sensitivity of these methods is not satisfactory for screening purpose. Recently, several new PCR-based mutation screening methods have been developed, but most of them require special instruments and adjustment of conditions for each DNA sequence to attain the maximum sensitivity, eventually becoming as inconvenient as old methods. Enzyme mismatch cleavage (EMC) is potentially an ideal screening method. With high-performance nucleases and once experimental conditions are optimized, it requires only conventional staff and conditions remain the same for each PCR product. In this study we tested four commercially available endonucleases for EMC and optimized the electrophoresis and developing conditions. We prepared 25 known DNA variations consisting of 18 single base substitutions (8 transitions and 10 transversions, including all possible sets of mismatches) and 7 small deletions or insertions. The combination of CEL nuclease, 12% PAGE and rapid silver staining can detect all types of mutations and achieved 100% sensitivity.

Amplification of padlock probes for DNA diagnostics by cascade rolling circle amplification or the polymerase chain reaction.

DC Thomas — 2008-07-19T03:25:43-00:00

Archives of pathology & laboratory medicine, Vol. 123, No. 12. (December 1999), pp. 1170-1176.

CONTEXT: Padlock probes are highly specific reagents for DNA diagnostics that can discriminate gene sequences with single base mutations. When the 3' and 5' terminal regions of the oligonucleotide probes are juxtaposed on a target DNA sequence, they can be circularized by enzymatic ligation and become topologically locked to the target. However, to be useful in solution-based diagnostics, the sensitivity of padlock probes must be markedly enhanced. OBJECTIVE: To describe two methods for geometric amplification of circularized padlock probes. DESIGN: Cascade rolling circle amplification is an isothermal system that uses generic primers and a DNA polymerase with strong strand displacement activity to amplify circularized probes by a mechanism combining rolling circle replication and strand displacement synthesis. One of the primers was designed as an energy transfer-labeled primer, which generates a fluorescence signal only when incorporated into the amplified product, enabling a direct means of detection. RESULTS: Using pUC19 as a model target to circularize an 89-base probe, a 10 billion-fold amplification was achieved with Bst DNA polymerase (large fragment) within 1 hour starting with as few as 10 probe molecules. The polymerase chain reaction was also used to amplify ligated padlock probes in a rare target detection system. In mixing experiments containing both normal and mutant p53 or c-Ki-ras2 gene target sequences, mutant targets were easily detected in the presence of a 500-fold excess of normal target copies. CONCLUSION: These results indicate that padlock probes can be amplified to the high levels required for solution-based DNA diagnostics.

Mutation detection and single-molecule counting using isothermal rolling-circle amplification.

PM Lizardi — 2008-07-19T03:19:36-00:00

Nature genetics, Vol. 19, No. 3. (July 1998), pp. 225-232.

Rolling-circle amplification (RCA) driven by DNA polymerase can replicate circularized oligonucleotide probes with either linear or geometric kinetics under isothermal conditions. In the presence of two primers, one hybridizing to the + strand, and the other, to the - strand of DNA, a complex pattern of DNA strand displacement ensues that generates 10(9) or more copies of each circle in 90 minutes, enabling detection of point mutations in human genomic DNA. Using a single primer, RCA generates hundreds of tandemly linked copies of a covalently closed circle in a few minutes. If matrix-associated, the DNA product remains bound at the site of synthesis, where it may be tagged, condensed and imaged as a point light source. Linear oligonucleotide probes bound covalently on a glass surface can generate RCA signals, the colour of which indicates the allele status of the target, depending on the outcome of specific, target-directed ligation events. As RCA permits millions of individual probe molecules to be counted and sorted using colour codes, it is particularly amenable for the analysis of rare somatic mutations. RCA also shows promise for the detection of padlock probes bound to single-copy genes in cytological preparations.

Integration of DNA ligation and rolling circle amplification for the homogeneous, end-point detection of single nucleotide polymorphisms.

J Pickering — 2008-07-19T03:00:37-00:00

Nucleic acids research, Vol. 30, No. 12. (15 June 2002)

Association studies using common sequence variants or single nucleotide polymorphisms (SNPs) may provide a powerful approach to dissect the genetic inheritance of common complex traits. Such studies necessitate the development of cost-effective, high throughput technologies for scoring SNPs. The method described in this paper for the co-detection of both alleles of a SNP in a single homogeneous reaction combines the specificity of a high fidelity DNA ligation step with the power of rolling circle amplification. The incorporation of Amplifluor energy transfer primers enables signal detection in a homogeneous format, making this approach highly amenable to automation. The adaptation of the genotyping method for high throughput screening using conventional liquid handling systems is described.

Highly efficient somatic-mutation identification using Escherichia coli mismatch-repair detection

Brock Peters — 2007-09-01T06:04:57-00:00

Nature Methods, Vol. 4, No. 9. (19 August 2007), pp. 713-715.

SNP-specific array-based allele-specific expression analysis.

Hans T Bjornsson — 2008-03-31T08:09:11-00:00

Genome Res (27 March 2008)

We have developed an optimized array-based approach for customizable allele-specific gene expression (ASE) analysis. The central features of the approach are the ability to select SNPs at will for detection, and the absence of need to PCR amplify the target. A surprisingly long probe length (39-49 nt) was needed for allelic discrimination. Reconstitution experiments demonstrate linearity of ASE over a broad range. Using this approach, we have discovered at least two novel imprinted genes, NLRP2, which encodes a member of the inflammasome, and OSBPL1A, which encodes a presumed oxysterol-binding protein, were both preferentially expressed from the maternal allele. In contrast, ERAP2, which encodes an aminopeptidase, did not show preferential parent-of-origin expression, but rather, cis-acting nonimprinted differential allelic control. The approach is scalable to the whole genome and can be used for discovery of functional epigenetic modifications in patient samples.

In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes

Chatarina Larsson — 2004-12-06T02:31:56-00:00

Nature Methods, Vol. 1, No. 3. (18 November 2004), 227.

Predicting Functional Regulatory Polymorphisms.

Ali Torkamani — 2008-07-18T05:23:55-00:00

Bioinformatics (Oxford, England) (18 June 2008)

MOTIVATION: Limited availability of data has hindered the development of algorithms that can identify functionally meaningful regulatory single nucleotide polymorphisms (rSNPs). Given the large number of common polymorphisms known to reside in the human genome, the identification of functional rSNPs via laboratory assays will be costly and time-consuming. Therefore appropriate bioinformatics strategies for predicting functional rSNPs are necessary. Recent data from the ENCODE Project has significantly expanded the amount of available functional information relevant to noncoding regions of the genome, and, importantly, led to the conclusion that many functional elements in the human genome are not conserved. RESULTS: In this manuscript we describe how ENCODE data can be leveraged to probabilistically determine the functional and phenotypic significance of noncoding SNPs (ncSNPs). The method achieves excellent sensitivity ( approximately 80%) and specificity ( approximately 99%) based on a set of known phenotypically-relevant and non-functional SNPs. In addition, we show that our method is not overtrained through the use of cross-validation analyses. AVAILABILITY: The software platforms used in our analyses are freely available (http://www.cs.waikato.ac.nz/ml/weka/). In addition, we provide the training dataset (Supplemental Table 3), and our predictions (Supplemental Table 6), in the supplementary material.

Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions: Current and developing technologies.

Brian N Chorley — 2008-07-18T05:24:03-00:00

Mutation research (4 May 2008)

The most common form of genetic variation, single nucleotide polymorphisms or SNPs, can affect the way an individual responds to the environment and modify disease risk. Although most of the millions of SNPs have little or no effect on gene regulation and protein activity, there are many circumstances where base changes can have deleterious effects. Non-synonymous SNPs that result in amino acid changes in proteins have been studied because of their obvious impact on protein activity. It is well known that SNPs within regulatory regions of the genome can result in disregulation of gene transcription. However, the impact of SNPs located in putative regulatory regions, or rSNPs, is harder to predict for two primary reasons. First, the mechanistic roles of non-coding genomic sequence remain poorly defined. Second, experimental validation of the functional consequences of rSNPs is often slow and laborious. In this review, we summarize traditional and novel methodologies for candidate rSNPs selection, in particular in silico techniques that aid in candidate rSNP selection. Additionally we will discuss molecular biological techniques that assess the impact of rSNPs on binding of regulatory machinery, as well as functional consequences on transcription. Standard techniques such as EMSA and luciferase reporter constructs are still widely used to assess effects of rSNPs on binding and gene transcription; however, these protocols are often bottlenecks in the discovery process. Therefore, we highlight novel and developing high-throughput protocols that promise to aid in shortening the process of rSNP validation. Given the large amount of genomic information generated from a multitude of re-sequencing and genome-wide SNP array efforts, future focus should be to develop validation techniques that will allow greater understanding of the impact these polymorphisms have on human health and disease.

A universal algorithm for de novo decrypting of heterozygous indel sequences: A tool for personalized medicine.

Ching-Wan Lam — 2007-12-30T22:22:29-00:00

Clin Chim Acta (23 November 2007)

INTRODUCTION: Indels (insertions/deletions) are important DNA sequence variations because of the high frequency in the human genome, the deleterious effects on the reading frame and protein expression, and the association with disease and disease susceptibility of common diseases. In a recent study with a human individual with the whole genome sequenced, 292,102 heterozygous indels and 559,473 homozygous indels were identified. Decrypting such a large number of heterozygous indels is computationally intensive and requires efficient algorithms. However, the current algorithms for decrypting heterozygous indel cannot be applied to unprecedented sequenced genomes and cannot be performed without reference sequences or reference sequence tracings for sequenced genomes. METHODS: A new algorithm for de novo decrypting of heterozygous indels is conceptualized in the direction of isolating the indel sequence from the genotype or diploid sequence. A universal algorithm is described, here, for heterozygous indel detection, indel size determination, and de novo decrypting of the indel sequence without subtracting the diploid DNA sequence from the reference sequence or reference sequence tracing. RESULTS: The result obtained by this algorithm is exactly the same as that obtained by PolyPhred and PolyScan. Unlike these algorithms, this new algorithm is not computationally intense for large indels, is independent of sequencing technologies and applies to genotype data derived from all existing sequencing technology platforms. A read of only 29 bases is enough to reduce the false detection rate (FDR) to 1 in a million. CONCLUSIONS: This algorithm is unique amongst all the existing algorithms in terms of performing the task of indel detection, size determination, and decrypting simultaneously. This universal approach eliminates the requirement of a reference sequence or sequence tracing and makes this algorithm unique in decrypting unprecedented sequenced genomes. Because of the high frequency of heterozygous indels in human genome, this universal algorithm will greatly reduce the time required for post-sequencing data analysis in whole genome sequencing of an individual for the practice of personalized medicine.

Simple detection of large InDeLS by DHPLC: the ACE gene as a model.

RG Koyama — 2008-07-18T04:54:56-00:00

Journal of biomedicine & biotechnology, Vol. 2008 (2008)

Insertion-deletion polymorphism (InDeL) is the second most frequent type of genetic variation in the human genome. For the detection of large InDeLs, researchers usually resort to either PCR gel analysis or RFLP, but these are time consuming and dependent on human interpretation. Therefore, a more efficient method for genotyping this kind of genetic variation is needed. In this report, we describe a method that can detect large InDeLs by DHPLC (denaturating high-performance liquid chromatography) using the angiotensin-converting enzyme (ACE) gene I/D polymorphism as a model. The InDeL targeted in this study is characterized by a 288 bp Alu element insertion (I). We used DHPLC at nondenaturating conditions to analyze the PCR product with a flow through the chromatographic column under two different gradients based on the differences between D and I sequences. The analysis described is quick and easy, making this technique a suitable and efficient means for DHPLC users to screen InDeLs in genetic epidemiological studies.

Real-time PCR genotyping of aldehyde dehydrogenase-2 using displacing probes.

Y Gu — 2008-07-18T04:53:52-00:00

Clinical biochemistry, Vol. 40, No. 16-17. (November 2007), pp. 1325-1327.

OBJECTIVES: This study aimed to develop and validate a rapid, accurate method for aldehyde dehydrogenase-2 gene (ALDH2) genotyping. DESIGN AND METHODS: We reported a single-tube, real-time PCR method for ALDH2 allele detection using two displacing probes. RESULTS: The genotyping results of 136 human genomic DNA samples indicated 100% concordance between real-time PCR method and the PCR-RFLP analysis. CONCLUSIONS: The developed method could be used for routine clinical testing and high throughput genetic screening of ALDH2.

Genotyping of the angiotensin I-converting enzyme gene insertion/deletion polymorphism by the TaqMan method.

W Koch — 2008-07-18T04:51:32-00:00

Clinical chemistry, Vol. 51, No. 8. (August 2005), pp. 1547-1549.

BlastDigester--a web-based program for efficient CAPS marker design.

K Ilic — 2008-07-18T03:20:07-00:00

Trends in genetics : TIG, Vol. 20, No. 7. (July 2004), pp. 280-283.

SNP2CAPS: a SNP and INDEL analysis tool for CAPS marker development.

T Thiel — 2008-07-17T16:31:22-00:00

Nucleic acids research, Vol. 32, No. 1. (2004)

With the influx of various SNP genotyping assays in recent years, there has been a need for an assay that is robust, yet cost effective, and could be performed using standard gel-based procedures. In this context, CAPS markers have been shown to meet these criteria. However, converting SNPs to CAPS markers can be a difficult process if done manually. In order to address this problem, we describe a computer program, SNP2CAPS, that facilitates the computational conversion of SNP markers into CAPS markers. 413 multiple aligned sequences derived from barley ESTs were analysed for the presence of polymorphisms in 235 distinct restriction sites. 282 (90%) of 314 alignments that contain sequence variation due to SNPs and InDels revealed at least one polymorphic restriction site. After reducing the number of restriction enzymes from 235 to 10, 31% of the polymorphic sites could still be detected. In order to demonstrate the usefulness of this tool for marker development, we experimentally validated some of the results predicted by SNP2CAPS.

DigiTag assay for multiplex single nucleotide polymorphism typing with high success rate.

N Nishida — 2008-07-17T14:53:25-00:00

Analytical biochemistry, Vol. 346, No. 2. (15 November 2005), pp. 281-288.

As a consequence of Human Genome Project and single nucleotide polymorphism (SNP) discovery projects, several millions of SNPs, which include possible susceptibility SNPs for multifactorial diseases, have been revealed. Accordingly, there has been a strong drive to perform the investigation with all candidate SNPs for a certain disease without decreasing the number of analyzed SNPs. We developed DigiTag assay, which uses well-designed oligonucleotides called DNA coded numbers (DCNs) in multiplex SNP genotype analysis. During the analysis, the information of a genotype is converted to one of the DCNs in a one to one manner using oligonucleotide ligation assay (encoding). After the encoding reaction, only the DCNs regions and not the SNP specific regions are amplified using the universal primers and then SNP genotype is read out using DNA capillary arrays. DigiTag assay was found to be successful in SNP genotyping, giving a high success rate (24 of 27 SNPs) for randomly chosen SNPs. Moreover, this assay has the potential to analyze almost all kinds of the target SNPs by applying mismatch-induced probes and redesigned primer pairs at a low-cost.

Amplification of circularizable probes for the detection of target nucleic acids and proteins.

D Zhang — 2008-07-17T13:49:46-00:00

Clinica chimica acta; international journal of clinical chemistry, Vol. 363, No. 1-2. (January 2006), pp. 61-70.

BACKGROUND: Circularizable oligonucleotide probe (C-probe) is a unique molecule that offers significant advantages over conventional probes. METHODS: Closed circular structure can be formed through ligation of the juxtaposed ends of the C-probe after hybridization with a target, and subsequently locked onto its target through the helical turns formed between the complementary sequences of the target and the C-probe (padlock probe). Under isothermal condition, C-probe can be amplified by rolling circle amplification (RCA) to generate multimeric single-stranded DNA (ssDNA). This multimeric ssDNA can be further amplified by a ramification mechanism (RAM) through primer extension and downstream DNA displacement, resulting in an exponential amplification. Usually, an unbiased product is generated by either RCA or ramification amplification method (or RAM) due to the generic primers of C-probe and its localization onto DNA targets. CONCLUSIONS: These advantages make C-probe amplification very useful for research and molecular diagnosis, especially in areas where other techniques were proved to be inadequate. The development of C-probe-based technologies offers a promising prospect for molecular diagnosis. The applications of C-probe, RCA, RAM, in situ detection, microarray, immunoassay, single nucleotide polymorphism, and whole genome amplification, etc. are discussed in this review.

Homogeneous and label-free bioluminescence detection of single nucleotide polymorphism with rolling circle amplification.

Y Cheng — 2008-07-17T13:15:19-00:00

The Analyst, Vol. 133, No. 6. (June 2008), pp. 750-752.

Integration of rolling circle amplification and sensitive bioluminescent detection of pyrophosphate, a homogeneous and label-free method has been developed for detecting single nucleotide polymorphism.

PCR primer design using statistical modeling.

A Yuryev — 2008-07-17T08:42:01-00:00

Methods in molecular biology (Clifton, N.J.), Vol. 402 (2007), pp. 93-104.

I describe the approaches for choosing primer parameters and calculating primer properties to build a statistical model for PCR primer design. Statistical modeling allows you to fine-tune the PCR primer design for your standard PCR conditions. It is most appropriate for the large organizations routinely performing PCR on the large scale or for the instruments that utilize PCR. This chapter shows how to use the statistical model to optimize the PCR primer design and to cluster primers for multiplex PCR. These methods have been developed to optimize single-nucleotide polymorphism-identification technology (SNP-IT) reaction for SNP genotyping and implemented in the Autoprimer program (http://www.autoprimer.com). The approaches for combining the individual primer scores into statistical model are described in the next chapter.

Fast masking of repeated primer binding sites in eukaryotic genomes.

R Andreson — 2008-07-17T08:39:30-00:00

Methods in molecular biology (Clifton, N.J.), Vol. 402 (2007), pp. 201-218.

In this article, we describe the working principle and a list of practical applications for GenomeMasker-a program that finds and masks all repeated DNA motifs in fully sequenced genomes. The GenomeMasker exhaustively finds and masks all repeated DNA motifs in studied genomes. The software is optimized for polymerase chain reaction (PCR) primer design. The algorithm is designed for high-throughput work, allowing masking of large DNA regions, even entire eukaryotic genomes. Additionally, the software is able to predict all alternative PCR products from studied genomes for thousands of candidate PCR primer pairs. Practical applications of the GenomeMasker are shown for command-line version of the GenomeMasker, which can be downloaded from http://bioinfo.ut.ee/download/. Graphical Web interfaces with limited options are available at http://bioinfo.ut.ee/genometester/ and http://bioinfo.ut.ee/snpmasker/.

Quantitative Single-letter Sequencing: a method for simultaneously monitoring numerous known allelic variants in single DNA samples

Baptiste Monsion — 2008-02-23T02:10:04-00:00

BMC Genomics, Vol. 9 (21 February 2008), 85.

FIT probes: peptide nucleic acid probes with a fluorescent base surrogate enable real-time DNA quantification and single nucleotide polymorphism discovery.

E Socher — 2008-07-17T08:30:48-00:00

Analytical biochemistry, Vol. 375, No. 2. (15 April 2008), pp. 318-330.

The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA-PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA-PNA duplexes. The technique employs so-called FIT (forced intercalation) probes in which one base is replaced by a thiazole orange (TO) dye molecule. If a DNA molecule that is complementary to the FIT-PNA molecule (except at the site of the dye) hybridizes to the probe, the TO dye exhibits intense fluorescence because stacking in the duplexes enforces a coplanar arrangement even in the excited state. However, a base mismatch at either position immediately adjacent to the TO dye dramatically decreases fluorescence, presumably because the TO dye has room to undergo torsional motions that lead to rapid depletion of the excited state. Of note, we found that the use of d-ornithine rather than aminoethylglycine as the PNA backbone increases the intensity of fluorescence emitted by matched probe-target duplexes while specificity of fluorescence signaling under nonstringent conditions is also increased. The usefulness of the ornithine-containing FIT probes was demonstrated in the real-time PCR analysis providing a linear measurement range over at least seven orders of magnitude. The analysis of two important single nucleotide polymorphisms (SNPs) in the CFTR gene confirmed the ability of FIT probes to facilitate unambiguous SNP calls for genomic DNA by quantitative PCR.

The LightTyper: high-throughput genotyping using fluorescent melting curve analysis.

CD Bennett — 2008-07-16T16:19:07-00:00

BioTechniques, Vol. 34, No. 6. (June 2003)

Instrumentation, chemistry, and software for high-throughput genotyping using fluorescent melting curves are described. The LightTyper system provides post-amplification genotyping within 10 min using samples in 96- or 384-well microplate formats. The system is homogenous because all reagents are added at the beginning of the reaction and there is no sample manipulation between amplification and genotyping. High-resolution melting curves are achieved by slow and steady heating. As samples are heated, panels of blue light-emitting diodes excite the probes, and fluorescence emission is acquired with a cooled charge-coupled device camera. A variety of probe chemistries are compatible for genotyping on the LightTyper, including dsDNA dyes, single-labeled probes, and fluorescence resonance energy transfer systems. Genotyping is performed automatically, and each sample is given a score reflecting the similarity of the genotype to the standards provided. Standard genotypes can be selected from within the run or imported from other files. Samples and genotypes can be grouped to allow multiple-allele detection on one or many samples. The utility of the LightTyper is illustrated by genotyping samples for the Factor V Leiden mutation and for mutations in the CFTR gene.

Hexaprimer Amplification Refractory Mutation System PCR for Simultaneous Single-Tube Genotyping of 2 Close Polymorphisms

Patrizia Piccioli — 2008-07-16T15:41:26-00:00

Clin Chem, Vol. 54, No. 1. (1 January 2008), pp. 227-229.

10.1373/clinchem.2007.095703

Closed-tube genotyping of apolipoprotein E by isolated-probe PCR with multiple unlabeled probes and high-resolution DNA melting analysis.

MD Poulson — 2008-07-16T15:40:49-00:00

BioTechniques, Vol. 43, No. 1. (July 2007), pp. 87-91.

Isolated-probe PCR (IP-PCR) is a method that combines asymmetric PCR, unlabeled probes, and high-resolution DNA melting while maintaining a closed tube system. A double-stranded DNA (dsDNA) dye LCGreen I was used to detect the unlabeled probes. LCGreen I is also used to detect the 277-base pair PCR product peak as an internal amplification control. To accomplish this, IP-PCR separates the asymmetric PCR amplification step and the detection step of the unlabeled probes. This prevents the probes from interfering with the amplification of the DNA target. The samples are then melted using a high-resolution DNA melting instrument: the HR-1. The closed tube system virtually eliminates PCR product contamination or sample carryover The target apolipoprotein E (APOE) was chosen to test the IP-PCR technique. APOE contains two single nucleotide polymorphisms (SNPs) located 139 base pairs apart in a GC-rich region of the human genome. The results from this study show that the IP-PCR technique was able to determine the correct APOE genotype for each of the 101 samples. The IP-PCR technique should also be useful in detecting SNPs in other high-GC regions of the human genome.

An optimal DNA pooling strategy for progressive fine mapping.

Xiao-Fei Chi — 2008-07-16T14:39:03-00:00

Genetica (28 May 2008)

We present a cost-effective DNA pooling strategy for fine mapping of a single Mendelian gene in controlled crosses. The theoretical argument suggests that it is potentially possible for a single-stage pooling approach to reduce the overall experimental expense considerably by balancing costs for genotyping and sample collection. Further, the genotyping burden can be reduced through multi-stage pooling. Numerical results are provided for practical guidelines. For example, the genotyping effort can be reduced to only a small fraction of that needed for individual genotyping at a small loss of estimation accuracy or at a cost of increasing sample sizes slightly when recombination rates are 0.5% or less. An optimal two-stage pooling scheme can reduce the amount of genotyping to 19.5%, 14.5% and 6.4% of individual genotyping efforts for identifying a gene within 1, 0.5, and 0.1 cM, respectively. Finally, we use a genetic data set for mapping the rice xl(t) gene to demonstrate the feasibility and efficiency of the DNA pooling strategy. Taken together, the results demonstrate that this DNA pooling strategy can greatly reduce the genotyping burden and the overall cost in fine mapping experiments.

Estimation of single-nucleotide polymorphism allele frequency by alternately binding probe competitive polymerase chain reaction.

N Noda — 2008-07-16T14:34:09-00:00

Analytica chimica acta, Vol. 608, No. 2. (11 February 2008), pp. 211-216.

Estimation of single-nucleotide polymorphism (SNP) allele frequency in pooled DNA samples is a promising approach to clarify the relationships between SNPs and diseases. Here, we present a simple, accurate, and cost-effective method for estimating SNP allele frequency, called alternately binding probe (ABProbe) competitive polymerase chain reaction (ABC-PCR) that entails no expensive devices for real-time fluorescence measurement and complex post-PCR steps. We prepared DNA pools of PCR products derived from homozygous samples of three different SNPs (ALDH2, GNB3, and HTR2A) in different portions, and the allele frequencies of these samples were estimated by ABC-PCR. Two alleles were coamplified by PCR with a fluorescent probe that binds to either alleles, and then fluorescence intensity was measured using a simple fluorometer. The ratio of the two alleles was calculated from the fluorescence intensity of the probe at the end-point. The estimated allele frequencies strongly correlated to the expected ratios for all three SNPs with high accuracy. When the allele frequencies were more than 5%, the relative standard deviations (R.S.D.s) of ABC-PCR were less than 20%. Moreover, we also confirmed that this method was applicable to SNP genotyping.

An alternative method for genotyping of the ACE I/D polymorphism.

Kimberly Glenn — 2008-07-16T13:14:08-00:00

Molecular biology reports (13 July 2008)

The mistyping of the angiotensin I-converting enzyme insertion/deletion (ACE I/D) has been well documented, and new methods have been suggested here to improve the genotyping efficiency. Buccal cell samples were collected from 157 young Caucasians, and genotyped using previously known and newly developed PCR amplification genotyping techniques, as well as PCR-RFLP tests for three single nucleotide polymorphisms (rs4327, rs4341 and rs4343). Inconsistent genotyping results were found when using only the PCR amplification genotyping techniques across repeated attempts (8% to 45%), however, individual SNP genotyping was highly consistent (100%). Two SNPs (rs4341 and rs4343) were in complete LD and SNP rs4327 was in high LD with the ACE I/D. The ACE I/D was in HW equilibrium in the portion of the population with consistent genotyping results, whereas the three SNPs were not in HW equilibrium. The mistyping of ACE I/D by only PCR amplification can be improved using alternative methods.

Homogeneous and label-free fluorescence detection of single-nucleotide polymorphism using target-primed branched rolling circle amplification.

Y Cheng — 2008-07-16T14:30:43-00:00

Analytical biochemistry, Vol. 378, No. 2. (15 July 2008), pp. 123-126.

We present a simple, sensitive, and cost-effective fluorescent assay of single-nucleotide polymorphism (SNP) with target-primed branched rolling circle amplification (TPBRCA). Designed padlock probe is circularized after perfect hybridization to mutant DNA. Then rolling circle amplification (RCA) reaction can be initiated from the mutant DNA that acts as primer and generates a long tandem single-stranded DNA (ssDNA) product. At the same time, the introduction of a reverse primer complementary to the target-primed RCA products leads to the branched RCA and eventually generates the various lengths of ssDNA and double-stranded DNA products, which are sensitively detected using SYBR Green I (SG) fluorescence dye. In contrast, the wild DNA contains a single mismatched base with the padlock probe and primes only a limited extension with the unligated padlock probe, generating weak background fluorescence with the addition of SG. Due to the excellent specificity and powerful amplification of TPBRCA reaction, the mutant DNA was distinctively differentiated from the wild DNA in a homogeneous and label-free manner. The assay is sensitive and specific enough to detect 5-amol (8.6-fM) mutant DNA strands. It was possible to accurately determine the mutant allele frequency as low as 1.0%.

A comprehensive assay for targeted multiplex amplification of human DNA sequences.

Sujatha Krishnakumar — 2008-07-16T13:40:08-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 27. (8 July 2008), pp. 9296-9301.

We developed a robust and reproducible methodology to amplify human sequences in parallel for use in downstream multiplexed sequence analyses. We call the methodology SMART (Spacer Multiplex Amplification Reaction), and it is based, in part, on padlock probe technology. As a proof of principle, we used SMART technology to simultaneously amplify 485 human exons ranging from 100 to 500 bp from human genomic DNA. In multiple repetitions, >90% of the targets were successfully amplified with a high degree of uniformity, with 70% of targets falling within a 10-fold range and all products falling within a 100-fold range of each other in abundance. We used long padlock probes (LPPs) >300 bases in length for the assay, and the increased length of these probes allowed for the capture of human sequences up to 500 bp in length, which is optimal for capturing most human exons. To engineer the LPPs, we developed a method that generates ssDNA molecules with precise ends, using an appropriately designed dsDNA template. The template has appropriate restriction sites engineered into it that can be digested to generate nucleotide overhangs that are suitable for lambda exonuclease digestion, producing a single-stranded probe from dsDNA. The SMART technology is flexible and can be easily adapted to multiplex tens of thousands of target sequences in a single reaction.

Mapping and sequencing of structural variation from eight human genomes

Jeffrey Kidd — 2008-04-30T19:31:59-00:00

Nature, Vol. 453, No. 7191., pp. 56-64.

Multiplex-Ready PCR: A new method for multiplexed SSR and SNP genotyping

Matthew Hayden — 2008-02-18T13:58:46-00:00

BMC Genomics, Vol. 9 (18 February 2008), 80.

PoooL: An efficient method for estimating haplotype frequencies from large DNA pools.

Han Zhang — 2008-07-16T13:29:51-00:00

Bioinformatics (Oxford, England) (23 June 2008)

MOTIVATION: Pooling DNA is a cost-effective alternative to individual genotyping method. It is often used for initial screening in genomewide association analysis. In some studies, large pools with sizes up to several hundreds were applied in order to significantly reduce genotyping cost. However, method for estimating haplotype frequencies from large DNA pools has not been available due to computational complexity involved. METHODS: We propose a novel constrained EM algorithm, PoooL, to estimate frequencies of single-nucleotide polymorphism (SNP) haplotypes from DNA pools. A quantity called importance factor is introduced to measure the contribution of a haplotype to the likelihood. Under the assumption of asymptotic normality of the estimated allele frequencies and a system of linear constraints on haplotype frequencies the importance factor remains a constant in the iterative maximization process. The maximization problem in the EM algorithm is then formulated into a constrained maximum entropy model and solved by the improved iterative scaling method. RESULTS: Simulation study shows that our algorithm can efficiently estimate haplotype frequencies from DNA pools with arbitrarily large sizes. The algorithm works equally well for large pools with sizes up to hundreds or thousands and for pools with sizes as small as one or two individuals. The computational complexity of the PoooL algorithm is independent of pool sizes, and the computational efficiency for large pools is thus substantially improved over existing estimating methods. Simulation results also show that the proposed method is robust to genotype errors and population admixture. AVAILABILITY: http://staff.ustc.edu.cn/~ynyang/poool CONTACT: zhanghan@mail.ustc.edu.cn, ynyang@ustc.edu.cn.

Individual single tube genotyping and DNA pooling by allele-specific PCR to uncover associations of polymorphisms with complex diseases.

A Casado-Díaz — 2008-07-16T13:26:36-00:00

Clinica chimica acta; international journal of clinical chemistry, Vol. 376, No. 1-2. (February 2007), pp. 155-162.

BACKGROUND: The genotyping for the study of the SNPs in different complex diseases require a great number of patients. In this sense, the determination of allele frequencies and genotypes requires a rapid and economical procedure. METHODS: The genotype has been carried out by allele-specific PCR in single tube with the discrimination of the products of PCR by its T(m). For this purpose a GC tail was added to 5' extreme of the specific primer. The allele frequencies were also calculated by DNA pooling and QRT-PCR using allele-specific primers. RESULTS: The use of the genotyping in single tube through allele-specific PCR and melting curves has led us to the accurate genotype of three polymorphisms of vdr (cdx-2), osteoprotegerin (A-163G) and ppar-gamma (C-681G) genes in 225 postmenopausal women to be associated to osteoporosis. Only the cdx-2 polymorphism was associated with a reduced bone mineral density (BMD). These data were similar to those obtained when the allele frequencies were calculated using QRT-PCR in DNA pools. CONCLUSIONS: Individual genotyping with allele-specific PCR in single tube and melting curve analysis is a fast, trustworthy and economic method to study any SNP. We propose the following approach to determine the possible association of SNPs with complex and multifactorial diseases like osteoporosis, in which hundreds of individuals should be analyzed: construct control and problem groups, make DNA pools, and calculate pooled allelic frequencies. Genotyping each individual further permits to determine the genotypic distribution when differences in allelic frequencies are observed, thus allowing more complex statistical analyses (including other variables like age, weight, etc.).

Development of a single tube 640-plex genotyping method for detection of nucleic acid variations on microarrays.

K Krjutskov — 2008-07-16T12:53:32-00:00

Nucleic acids research, Vol. 36, No. 12. (July 2008)

Detection of DNA sequence variation is critical to biomedical applications, including disease genetic identification, diagnosis and treatment, drug discovery and forensic analysis. Here, we describe an arrayed primer extension-based genotyping method (APEX-2) that allows multiplex (640-plex) DNA amplification and detection of single nucleotide polymorphisms (SNPs) and mutations on microarrays via four-color single-base primer extension. The founding principle of APEX-2 multiplex PCR requires two oligonucleotides per SNP/mutation to generate amplicons containing the position of interest. The same oligonucleotides are then subsequently used as immobilized single-base extension primers on a microarray. The method described here is ideal for SNP or mutation detection analysis, molecular diagnostics and forensic analysis. This robust genetic test has minimal requirements: two primers, two spots on the microarray and a low cost four-color detection system for the targeted site; and provides an advantageous alternative to high-density platforms and low-density detection systems.

Successful amplification of degraded DNA for use with high-throughput SNP genotyping platforms.

Simon Mead — 2008-07-16T12:44:40-00:00

Human mutation (12 June 2008)

Highly accurate and high-throughput SNP genotyping platforms are increasingly popular but the performance of suboptimal DNA samples remains unclear. The aim of our study was to determine the best platform, amplification technique, and loading concentration to maximize genotype accuracy and call rate using degraded samples. We amplified high-molecular weight genomic DNA samples recently extracted from whole blood and degraded DNA samples extracted from 50-year-old patient sera. Two whole-genome amplification (WGA) methodologies were used: an isothermal multiple displacement amplification method (MDA) and a fragmentation-PCR-based method (GenomePlex((R)) [GPLEX]; Sigma-Aldrich, St. Louis, MO). Duplicate runs were performed on genome-wide dense SNP arrays (Nsp-Mendel; Affymetrix) and custom SNP platforms based on molecular inversion probes (Targeted Genotyping [TG]; Affymetrix) and BeadArray technology (Golden Gate [GG]; Illumina). Miscalls and no-calls on Mendel arrays were correlated with each other, with confidence scores from the Bayesian calling algorithm, and with average probe intensity. Degraded DNA amplified with MDA gave low call rates and concordance across all platforms at standard loading concentrations. The call rate with MDA on GG was improved when a 5 x concentration of amplified DNA was used. The GPLEX amplification gave high call rate and concordance for degraded DNA at standard and higher loading concentrations on both TG and GG platforms. Based on these analyses, after standard filtering for SNP and sample performance, we were able to achieve a mean call rate of 99.7% and concordance 99.7% using degraded samples amplified by GPLEX on GG technology at 2 x loading concentration. These findings may be useful for investigators planning case-control association studies with patient samples of suboptimal quality. Hum Mutat 0, 1-7, 2008. (c) 2008 Wiley-Liss, Inc.

The Framingham Heart Study 100K SNP genome-wide association study resource: overview of 17 phenotype working group reports.

LA Cupples — 2008-04-16T16:12:34-00:00

BMC medical genetics, Vol. 8 Suppl 1 (2007)

BACKGROUND: The Framingham Heart Study (FHS), founded in 1948 to examine the epidemiology of cardiovascular disease, is among the most comprehensively characterized multi-generational studies in the world. Many collected phenotypes have substantial genetic contributors; yet most genetic determinants remain to be identified. Using single nucleotide polymorphisms (SNPs) from a 100K genome-wide scan, we examine the associations of common polymorphisms with phenotypic variation in this community-based cohort and provide a full-disclosure, web-based resource of results for future replication studies.METHODS: Adult participants (n = 1345) of the largest 310 pedigrees in the FHS, many biologically related, were genotyped with the 100K Affymetrix GeneChip. These genotypes were used to assess their contribution to 987 phenotypes collected in FHS over 56 years of follow up, including: cardiovascular risk factors and biomarkers; subclinical and clinical cardiovascular disease; cancer and longevity traits; and traits in pulmonary, sleep, neurology, renal, and bone domains. We conducted genome-wide variance components linkage and population-based and family-based association tests.RESULTS: The participants were white of European descent and from the FHS Original and Offspring Cohorts (examination 1 Offspring mean age 32 +/- 9 years, 54% women). This overview summarizes the methods, selected findings and limitations of the results presented in the accompanying series of 17 manuscripts. The presented association results are based on 70,897 autosomal SNPs meeting the following criteria: minor allele frequency > or + 10%, genotype call rate > or = 80%, Hardy-Weinberg equilibrium p-value > or = 0.001, and satisfying Mendelian consistency. Linkage analyses are based on 11,200 SNPs and short-tandem repeats. Results of phenotype-genotype linkages and associations for all autosomal SNPs are posted on the NCBI dbGaP website at http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007 webcite.CONCLUSION: We have created a full-disclosure resource of results, posted on the dbGaP website, from a genome-wide association study in the FHS. Because we used three analytical approaches to examine the association and linkage of 987 phenotypes with thousands of SNPs, our results must be considered hypothesis-generating and need to be replicated. Results from the FHS 100K project with NCBI web posting provides a resource for investigators to identify high priority findings for replication.

SNPAnalyzer 2.0: a web-based integrated workbench for linkage disequilibrium analysis and association analysis

Jinho Yoo — 2008-06-24T10:44:48-00:00

BMC Bioinformatics, Vol. 9 (23 June 2008), 290.