SNP500Cancer: a public resource for sequence validation, assay development, and frequency analysis for genetic variation in candidate genes.

The SNP500Cancer database provides sequence and genotype assay information for candidate SNPs useful in mapping complex diseases, such as cancer. The database is an integral component of the NCI Cancer Genome Anatomy Project (http://cgap.nci.nih.gov). SNP500Cancer reports sequence analysis of anonymized control DNA samples (n = 102 Coriell samples representing four self-described ethnic groups: African/African-American, Caucasian, Hispanic and Pacific Rim). The website is searchable by gene, chromosome, gene ontology pathway, dbSNP ID and SNP500Cancer SNP ID. As of October 2005, the database contains >13 400 SNPs, 9124 of which have been sequenced in the SNP500Cancer population. For each analysed SNP, gene location and >200 bp of surrounding annotated sequence (including nearby SNPs) are provided, with frequency information in total and per subpopulation as well as calculation of Hardy-Weinberg equilibrium for each subpopulation. The website provides the conditions for validated sequencing and genotyping assays, as well as genotype results for the 102 samples, in both viewable and downloadable formats. A subset of sequence validated SNPs with minor allele frequency >5% are entered into a high-throughput pipeline for genotyping analysis to determine concordance for the same 102 samples. In addition, the results of genotype analysis for select validated SNP assays (defined as 100% concordance between sequence analysis and genotype results) are posted for an additional 280 samples drawn from the Human Diversity Panel (HDP). SNP500Cancer provides an invaluable resource for investigators to select SNPs for analysis, design genotyping assays using validated sequence data, choose selected assays already validated on one or more genotyping platforms, and select reference standards for genotyping assays. The SNP500Cancer database is freely accessible via the web page at http://snp500cancer.nci.nih.gov.

Comparison of yield and genotyping performance of multiple displacement amplification and OmniPlex whole genome amplified DNA generated from multiple DNA sources.

The promise of whole genome amplification (WGA) is that genomic DNA (gDNA) quantity will not limit molecular genetic analyses. Multiple displacement amplification (MDA) and the OmniPlex PCR-based WGA protocols were evaluated using 4 and 5 ng of input gDNA from 60 gDNA samples from three tissue sources (mouthwash, buffy coat, and lymphoblast). WGA DNA (wgaDNA) yield and genotyping performance were evaluated using genotypes determined from gDNA and wgaDNA using the AmpFlSTR Identifiler assay and N = 49 TaqMan SNP assays. Short tandem repeat (STR) and SNP genotyping completion and concordance rates were significantly reduced with wgaDNA from all WGA methods compared with gDNA. OmniPlex wgaDNA exhibited a greater reduction in genotyping performance than MDA wgaDNA. Reduced wgaDNA genotyping performance was due to allelic (all protocols) and locus (OmniPlex) amplification bias leading to heterozygote and locus dropout, respectively, and %GC sequence content (%GC) was significantly correlated with TaqMan assay performance. Lymphoblast wgaDNA exhibited higher yield (OmniPlex), buffy coat wgaDNA exhibited higher STR genotyping completion (MDA), whereas mouthwash wgaDNA exhibited higher SNP genotyping discordance (MDA). Genotyping of wgaDNA generated from < or = 5 ng gDNA, e.g., from archaeological, forensic, prenatal diagnostic, or pathology samples, may require additional genotyping validation with gDNA and/or more sophisticated analysis of genotypes incorporating observed reductions in genotyping performance.

Corroboration of a familial chordoma locus on chromosome 7q and evidence of genetic heterogeneity using single nucleotide polymorphisms (SNPs).

Chordoma, a rare bone tumor originating from notochordal remnants, has a genetic predisposition in some families. Previously, we performed linkage analysis using microsatellite (STR) markers on 3 unrelated chordoma kindreds (16 patients with chordoma) and reported significant evidence for linkage to chromosome 7q33 (Z(max) = 4.78) with a minimal disease gene region of 11 cM. In our present study, we performed linkage analysis in these 3 families using chromosome 7 single nucleotide polymorphisms (SNPs). Parametric and nonparametric multipoint analyses showed significant linkage to 7q markers with p < 0.001 and Z(max) = 2.77, respectively. The minimal disease gene region was not reduced by combined SNP and STR haplotype analysis compared to the previous STR haplotype analysis alone. We genotyped members of a fourth chordoma family with SNP and STR markers for chromosome 7q and for 1p36, the location of a previously reported chordoma locus. Affected members of this family did not share a common haplotype on 7q, and the family did not show evidence of linkage to 1p36. Thus, we corroborated a chordoma locus on chromosome 7q in the 3 original families and demonstrated evidence for genetic heterogeneity in the fourth family. Our study also provided insights into some limitations and analytical complexities associated with using a dense SNP marker set in linkage analysis of complex pedigrees.

Controlled crystalline structure and surface stability of cobalt nanocrystals.

The synthesis of monodispersed 10 nm cobalt nanocrystals with controlled crystal morphology and investigation of the surface stability of these nanocrystals are described. Depending on the surfactants used, single crystalline or multiple grain nanocrystals can be reproducibly produced. The relative surface stability of these nanocrystals is analyzed using the temperature dependences of the dc magnetic susceptibility. The novel method, which allows sensitive monitoring of the surface stability, is based on the observation that, with particle oxidation, an anomalous peak appears at 8 K in zero-field-cooled magnetization measurements. It is found that the surfactant protective layer is more important for long-term stability at room temperature, while the high-temperature oxidation rate is controlled by the crystal morphology of the nanoparticles.

SNP500Cancer: a public resource for sequence validation and assay development for genetic variation in candidate genes.

The SNP500Cancer Database provides sequence and genotype assay information for candidate single nucleotide polymorphisms (SNPs) useful in mapping complex diseases, such as cancer. The database is an integral component of the NCI's Cancer Genome Anatomy Project. SNP500Cancer provides bi-directional sequencing information on a set of control DNA samples derived from anonymized subjects (102 Coriell samples representing four self-described ethnic groups: African/African-American, Caucasian, Hispanic and Pacific Rim). All SNPs are chosen from public databases and reports, and the choice of genes includes a bias towards non-synonymous and promoter SNPs in genes that have been implicated in one or more cancers. The web site is searchable by gene, chromosome, gene ontology pathway and by known dbSNP ID. As of July 2003, the database contains over 3400 SNPs, 2490 of which have been sequenced in the SNP500Cancer population. For each analyzed SNP, gene location and over 200 bp of surrounding annotated sequence (including nearby SNPs) are provided, with frequency information in total and per subpopulation, and calculation of Hardy-Weinberg Equilibrium (HWE) for each subpopulation. Sequence validated SNPs with minor allele frequency > 5% are entered into a high-throughput pipeline for genotyping analysis to determine concordance for the same 102 samples. The website provides the conditions for validated genotyping assays. SNP500Cancer provides an invaluable resource for investigators to select SNPs for analysis, design genotyping assays using validated sequence data, choose selected assays already validated on one or more genotyping platforms, and select reference standards for genotyping assays. The SNP500Cancer Database is freely accessible via the web page at http://snp500cancer.nci.nih.gov/.

Performance of high-throughput DNA quantification methods.

BACKGROUND: The accuracy and precision of estimates of DNA concentration are critical factors for efficient use of DNA samples in high-throughput genotype and sequence analyses. We evaluated the performance of spectrophotometric (OD) DNA quantification, and compared it to two fluorometric quantification methods, the PicoGreen assay (PG), and a novel real-time quantitative genomic PCR assay (QG) specific to a region at the human BRCA1 locus. Twenty-Two lymphoblastoid cell line DNA samples with an initial concentration of approximately 350 ng/uL were diluted to 20 ng/uL. DNA concentration was estimated by OD and further diluted to 5 ng/uL. The concentrations of multiple aliquots of the final dilution were measured by the OD, QG and PG methods. The effects of manual and robotic laboratory sample handling procedures on the estimates of DNA concentration were assessed using variance components analyses. RESULTS: The OD method was the DNA quantification method most concordant with the reference sample among the three methods evaluated. A large fraction of the total variance for all three methods (36.0-95.7%) was explained by sample-to-sample variation, whereas the amount of variance attributable to sample handling was small (0.8-17.5%). Residual error (3.2-59.4%), corresponding to un-modelled factors, contributed a greater extent to the total variation than the sample handling procedures. CONCLUSION: The application of a specific DNA quantification method to a particular molecular genetic laboratory protocol must take into account the accuracy and precision of the specific method, as well as the requirements of the experimental workflow with respect to sample volumes and throughput. While OD was the most concordant and precise DNA quantification method in this study, the information provided by the quantitative PCR assay regarding the suitability of DNA samples for PCR may be an essential factor for some protocols, despite the decreased concordance and precision of this method.

A genome-wide linkage scan for body mass index on Framingham Heart Study families.

BACKGROUND: Genome-wide scan data from a community-based sample was used to identify the genetic factors that affect body mass index (BMI). BMI was defined as weight (kg) over the square of height (m), where weight and height were obtained from the first measurement available between the ages of 40 and 50 years. RESULTS: Significant familial correlations were observed in mother:father (spouse) relative pairs and in all relative pairs examined except parent:daughter pairs. Single-point sib-pair regression analysis provided nominal evidence for linkage (p < 0.05) of loci to BMI at 23 markers. Multi-point sib-pair regression analysis provided nominal evidence for linkage to BMI at 42 loci on 12 chromosomes. Empirical p-values showed results consistent with the multi-point results; all but three of the loci identified by multi-point analysis were also significant. CONCLUSION: The largest regions of nominally significant linkage were found on chromosomes 2, 3, and 11. The most significant evidence for linkage was obtained with markers D2S1788, D2S1356, D2S1352, D3S1744, and D11S912 from multi-point sib-pair single-trait regression analysis. Our results are in agreement with some of the recently published reports on BMI using various data sets including the Framingham Heart Study data.

Genomic regions linked to alcohol consumption in the Framingham Heart Study.

BACKGROUND: Pedigree, demographic, square-root transformed maximum alcohol (SRMAXAPD) and maximum cigarette (MAXCPD) consumption, and genome-wide scan data from the Framingham Heart Study (FHS) were used to investigate genetic factors that may affect alcohol and cigarette consumption in this population-based sample. RESULTS: A significant sister:sister correlation greater than spouse correlation was observed for MAXCPD only. Single-point sib-pair regression analysis provided nominal evidence for linkage of loci to both SRMAXAPD and MAXCPD consumption traits, with more significant evidence of linkage to SRMAXAPD than to MAXCPD. One genomic region, chr9q21.11, exhibits significant multi-point sib-pair regression to SRMAXAPD. CONCLUSION: SRMAXAPD exhibits greater evidence for genetic linkage than does MAXCPD in the FHS sample. Four regions of the genome exhibiting nominal evidence for linkage to SRMAXAPD in the FHS sample correspond to regions of the genome previously identified as linked to alcoholism or related traits in the family data set ascertained on individuals affected with alcohol dependence known as COGA.