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PubMed2003

Linkage disequilibrium assessment via log-linear modeling of SNP haplotype frequencies.

Analyses of high-density single-nucleotide polymorphism (SNP) data, such as genetic mapping and linkage disequilibrium (LD) studies, require phase-known haplotypes to allow for the correlation between tightly linked loci. However, current SNP genotyping technology cannot determine phase, which must be inferred statistically. In this paper, we present a new Bayesian Markov chain Monte Carlo (MCMC) algorithm for population haplotype frequency estimation, particularly in the context of LD assessment. The novel feature of the method is the incorporation of a log-linear prior model for population haplotype frequencies. We present simulations to suggest that 1) the log-linear prior model is more appropriate than the standard coalescent process in the presence of recombination (>0.02 cM between adjacent loci), and 2) there is substantial inflation in measures of LD obtained by a "two-stage" approach to the analysis by treating the "best" haplotype configuration as correct, without regard to uncertainty in the recombination process.

Algorithms↗

PubMed2001

READIT: a novel technology used in the interrogation of nucleic acid sequences for single-nucleotide polymorphisms.

The ability to perform analyses for single-nucleotide polymorphisms (SNPs) has become routine in many molecular diagnostic laboratories. While various procedures and technologies are available to do so, we evaluated a novel technology for SNP analysis using the Factor V Leiden polymorphism as an example. The Factor V Leiden polymorphism G1691A is the most common genetic abnormality associated with hereditary thrombophilia. Current testing methods remain highly accurate yet their main disadvantage is gel-based detection. The READIT System (Promega Corp., Madison, WI) is a novel approach to SNP analysis that utilizes DNA polymerase-mediated pyrophosphorolysis to release dNTPs, which are converted to ATP and used in a luciferase detection reaction. We screened 280 DNA specimens, previously analyzed using the PCR/MnlI restriction digest assay, to evaluate READIT System capabilities along with the KingFisher robotic magnetic particle processor (ThermoLabsystems). Concordant results were obtained for 278/280 (99%) specimens. One discordant result was due to an equivocal relative light unit while the other was indeterminate. Both specimens gave correct results upon repeat analysis. The READIT System offers several advantages including: (1) rapid SNP analysis, (2) accuracy and precision, (3) cost effectiveness, (4) decreased turn-around times, (5) high throughput, and (6) excellent analysis software.

DNA↗

PubMed2003

Association and interaction of the IL4R, IL4, and IL13 loci with type 1 diabetes among Filipinos.

In the search for genes involved in type 1 diabetes (T1D), other than the well-established risk alleles at the human leukocyte antigen loci, we have investigated the association and interaction of polymorphisms in genes involved in the IL4/IL13 pathway in a sample of 90 Filipino patients with T1D and 94 controls. Ten single-nucleotide polymorphisms (SNPs), including two promoter SNPs in the IL4R locus on chromosome 16p11, one promoter SNP in the IL4 locus on chromosome 5q31, and four SNPs--including two promoter SNPs--in the IL13 locus on chromosome 5q31 were examined for association, linkage disequilibrium, and interaction. We found that both individual SNPs (IL4R L389L; odds ratio [OR] 0.34; 95% confidence interval [CI] 0.17-0.67; P=.001) and specific haplotypes both in IL4R (OR 0.10; 95% CI 0-0.5; P=.001) and for the five linked IL4 and IL13 SNPs (OR 3.47; P=.004) were strongly associated with susceptibility to T1D. Since IL4 and IL13 both serve as ligands for a receptor composed, in part, of the IL4R alpha chain, we looked for potential epistasis between polymorphisms in the IL4R locus on chromosome 16p11 and the five SNPs in the IL4 and IL13 loci on chromosome 5q31 and found, through use of a logistic-regression model, significant gene-gene interactions (P=.045, corrected for multiple comparisons by permutation analysis). Our data suggest that the risk for T1D is determined, in part, by polymorphisms within the IL4R locus, including promoter and coding-sequence variants, and by specific combinations of genotypes at the IL4R and the IL4 and IL13 loci.

Chromosomes, Human, Pair 16↗

PubMed2004

Genetic analysis of 22 candidate genes for hypertension in the Japanese population.

OBJECTIVE: We performed association studies between 118 single-nucleotide polymorphisms (SNPs) of 22 candidate genes (or gene family) and hypertension in a Japanese population. DESIGN AND PARTICIPANTS: The study population consisted of 1880 subjects representing the general population in Japan, recruited from the Suita study. The candidate genes were selected based on their functions, including insulin resistance (APM1, CD36, HSD11B1), oxidative stress (CYBA, GPX1, GSTMs), steroid hormone (ESR1, ESR2, HSD11B2), renal functions (PTGS2, KLK1, NPHS1, NPHS2, SGK, SLC12A1, PTGES), and others related to cardiovascular physiology (GJA4, NOS1, NTRK3, P2RX4, SPP1, ALDH2). RESULTS: Multiple logistic analyses, with age and body mass index as covariates, indicated that 13 SNPs (eight genes), six SNPs (four genes) and 11 SNPs (four genes) were associated with hypertension (P < 0.05) in the total, male, and female populations, respectively. PTGS2 seems to be a promising candidate gene for hypertension in men. GSTM3 and SLC12A1 seem to be promising candidate genes for hypertension in women. Especially, a polymorphism in SLC12A1 was significantly associated with hypertension in women even after correction by the Bonferroni method (corrected P = 0.0236). Multiple logistic analyses, with age and body mass index as covariates, indicated that the prevalence of hypertension in females was significantly higher in subjects with the CC genotype than in those with the TT + TC genotypes (P < 0.0001, odds ratio = 1.967, 95% confidence interval = 1.430-2.712). CONCLUSION: Although the present results should be replicated in other study populations for confirmation, the present results suggest that SLC12A1 may contribute to hypertension in Japanese women.

Adult↗

PubMed2002

Determination of the factor V Leiden single-nucleotide polymorphism in a commercial clinical laboratory by use of NanoChip microelectronic array technology.

BACKGROUND: Methods for analysis of the single-nucleotide polymorphism (SNP) known as factor V Leiden (FVL) are described. The technique provides rapid, highly accurate detection of the point mutation that encodes for replacement of arginine-506 with glutamine. After formal assay qualification, 758 clinical samples that had previously been analyzed by the Invader Monoplex Assay were tested as research samples in a commercial clinical laboratory. METHODS: Primers specific for factor V (FV) were prepared, and PCR was performed. Samples were analyzed using the NanoChip Molecular Biology Workstation with fluorescently labeled reporters for wild-type and SNP sequences. RESULTS: Of the 635 samples classified by the Third Wave assay as FV wild type, 10 were identified as heterozygous FVL by the NanoChip technique. Similarly, of the 114 putative heterozygous samples, 4 were wild type, and of the 9 reported homozygous samples, 6 were homozygous, 2 were heterozygous, and 1 was FV wild type by the NanoChip assay. All 17 results that were discordant with the Third Wave analysis were confirmed by DNA sequencing to be correctly classified by the NanoChip technology. The Nanochip system was 100% accurate in characterizing wild-type, heterozygous, and homozygous samples compared with accuracies of 99.2%, 90.2%, and 100% for the comparable Third Wave analysis. CONCLUSIONS: The NanoChip microelectronic chip array technology is an accurate and convenient method for FVL screening of research samples in a clinical laboratory environment.

Autoanalysis↗

PubMed2004

The allele frequency spectrum in genome-wide human variation data reveals signals of differential demographic history in three large world populations.

We have studied a genome-wide set of single-nucleotide polymorphism (SNP) allele frequency measures for African-American, East Asian, and European-American samples. For this analysis we derived a simple, closed mathematical formulation for the spectrum of expected allele frequencies when the sampled populations have experienced nonstationary demographic histories. The direct calculation generates the spectrum orders of magnitude faster than coalescent simulations do and allows us to generate spectra for a large number of alternative histories on a multidimensional parameter grid. Model-fitting experiments using this grid reveal significant population-specific differences among the demographic histories that best describe the observed allele frequency spectra. European and Asian spectra show a bottleneck-shaped history: a reduction of effective population size in the past followed by a recent phase of size recovery. In contrast, the African-American spectrum shows a history of moderate but uninterrupted population expansion. These differences are expected to have profound consequences for the design of medical association studies. The analytical methods developed for this study, i.e., a closed mathematical formulation for the allele frequency spectrum, correcting the ascertainment bias introduced by shallow SNP sampling, and dealing with variable sample sizes provide a general framework for the analysis of public variation data.

Black or African American↗

PubMed2004

Analysis of single-nucleotide polymorphisms in Japanese rheumatoid arthritis patients shows additional susceptibility markers besides the classic shared epitope susceptibility sequences.

OBJECTIVE: To examine the entire HLA region for loci (other than the DRB1 locus) associated with rheumatoid arthritis (RA) susceptibility, by typing HLA-DRB1 alleles and multiple single-nucleotide polymorphisms (SNPs) in the Japanese population. METHODS: The HLA-DRB1 alleles and 88 SNPs distributed over the HLA gene complex were genotyped, for 828 patients with RA and 1,032 control subjects. The data were evaluated for linkage disequilibrium, and case-control associations were analyzed in 2 ways, in the presence or absence of the disease-susceptibility DRB1 allele, to detect loci independent of the DRB1 allele. RESULTS: HLA-DRB1 alleles *0405, *0401, *0901, *0101, *1401, *1602, *0403, and *1405 were significantly associated with RA in the Japanese population. The smallest P value (P = 1.4 x 10(-27)) was observed in association with an intronic SNP of the NOTCH4 gene, which was due to strong linkage disequilibrium with the HLA-DRB1 allele. A strong association that was independent of HLA-DRB1 shared epitope alleles was observed in 2 SNPs: one in the intron of the MICA gene, the other in the intron of the HLA-DQB2 gene. Their association with RA, independent of HLA-DRB1 shared epitope alleles, was suggestive (P = 0.0024 [corrected P (P(corr)) = 0.068, and P = 0.00037 [P(corr) = 0.012], respectively). CONCLUSION: These findings suggest that 1 or more other loci besides the HLA-DRB1 or other DRB1 (non-shared epitope, non-*0901) alleles are involved in RA susceptibility/protection.

Adult↗

PubMed2026

No receptor-binding domain adaptation detected in within-host H5N1 surveillance of 4,559 US dairy outbreak sequences.

BACKGROUND: The 2024-2026 US H5N1 clade 2.3.4.4b dairy cattle outbreak has been characterised primarily through consensus-level phylogenetics. Whether mammalian-adaptation variants are emerging at sub-consensus frequencies within infected hosts, particularly at the haemagglutinin receptor-binding domain (RBD), remains unknown because no systematic within-host variant analysis of the public sequencing corpus has been performed. METHODS: We conducted a pre-registered, corpus-wide intrahost single-nucleotide variant (iSNV) analysis of all publicly available H5N1 cattle, feline-spillover, and retail-milk sequences on the NCBI Sequence Read Archive (4559 samples across 7 BioProjects). A dual-caller concordance pipeline (iVar + LoFreq) with empirically determined allele frequency (AF) threshold (3%, set via four-criterion validation including synthetic spike-in controls) was applied to an 11-site Tier 1 mammalian-adaptation panel spanning the polymerase complex, haemagglutinin RBD, and accessory proteins. Within-host nucleotide diversity was compared across host categories. RESULTS: The HA RBD sites Q226L and G228S (H3 numbering) showed zero detections across >4300 adequately sequenced samples at all AF thresholds tested (1-5%), despite the pipeline detecting other non-synonymous variants at these exact codon positions (upper 95% CI for prevalence: 0.08%). Seven of eleven adaptation sites carried statistically significant iSNV signals after Bonferroni correction (corrected α = 0.00417), though all at low prevalence (≤2.95%). Genotype stratification showed that most polymerase-site detections reflected genotype structure rather than within-host emergence: the apparent PB2 631 L→M "reversion" was largely the ancestral avian state of the D1.1 genotype (20 of 23 detections), which never acquired the 631L mammalian adaptation, with only two genuine sub-consensus events in the B3.13 background, while consensus-level PB2 701N was a fixed feature of the D1.1 genotype (10 of 14 detections) rather than independent sub-consensus emergence. Cattle exhibited significantly higher within-host nucleotide diversity than feline-spillover samples (π = 1.59 × 10-4 vs 6.11 × 10-5; Kruskal-Wallis p = 6.6 × 10-15), a finding that persisted after depth-matching (p = 4.6 × 10-5); this may reflect prolonged mammary-gland infection, though sampling differences and host biology cannot be excluded. CONCLUSIONS: We did not detect HA receptor-switching adaptation (the acquisition of human-type α2,6 receptor binding via Q226L/G228S) at any tested allele frequency in the US dairy H5N1 outbreak. Sub-consensus mammalian-adaptation signals exist at polymerase-complex sites but at low prevalence, are genotype-structured rather than independently recurrent, and require functional characterisation before informing risk assessment.

Dairy cattle↗

PubMed2001

A high-throughput SNP typing system for genome-wide association studies.

One of the most difficult issues to be solved in genome-wide association studies is to reduce the amount of genomic DNA required for genotyping. Currently available technologies require too large a quantity of genomic DNA to genotype with hundreds or thousands of single-nucleotide polymorphisms (SNPs). To overcome this problem, we combined the Invader assay with multiplex polymerase chain reaction (PCR), carried out in the presence of antibody to Taq polymerase, as well as using a novel 384-well card system that can reduce the required reaction volume. We amplified 100 genomic DNA fragments, each containing one SNP, in a single tube, and analyzed each SNP with the Invader assay. This procedure correctly genotyped 98 of the 100 SNP loci examined in PCR-amplified samples from ten individuals: the genotypes were confirmed by direct sequencing. The reproducibility and universality of the method were confirmed with two additional sets of 100 SNPs. Because we used 40 ng of genomic DNA as a template for multiplex PCR, the amount needed to assay one SNP was only 0.4 ng; therefore, theoretically, more than 200,000 SNPs could be genotyped at once when 100 microg of genomic DNA is available. Our results indicate the feasibility of undertaking genome-wide association studies using blood samples of only 5-10 ml.

Alleles↗

PubMed2026

SeqQC-former: A sequence-quality fusion framework for QC-aware review prioritization of candidate somatic SNVs in cancer genomics.

The accurate prioritization of candidate somatic single-nucleotide variants (SNVs) remains a challenge due to the substantial variability in sequencing quality across genomic loci. SeqQC-Former is a sequence-quality fusion framework that integrates the local nucleotide context with read-level quality-control (QC) covariates derived from matched tumor-normal sequencing data. This integration generates QC-aware prioritization scores for the downstream review of candidate variants. Unlike conventional variant callers, SeqQC-Former is designed not to infer biological truth but to support post-calling review and prioritization under heterogeneous sequencing conditions. The framework was trained and evaluated on a SEQC2-derived dataset comprising 89,447 candidate loci, including 1378 positive and 88,069 negative loci. In chromosome-held-out validation, which aims to reduce potential genomic-position leakage, SeqQC-Former demonstrated strong discrimination (AUROC = 0.9479; AUPRC = 0.9448), indicating good generalization to previously unseen chromosomes. Given that the SEQC2-derived labels contain QC-associated information; these results should be interpreted as an evaluation of QC-aware prioritization capability rather than an independent validation of biological variant correctness. Ablation analyses revealed that structured QC covariates provided the dominant predictive signal under the current SEQC2-derived labeling regime. SeqQC-Former achieved a significantly higher AUROC than classical machine-learning baselines, as determined by DeLong's test (p < 0.01). Application to 53,164 glioblastoma variants demonstrated that external predictions were sensitive to QC scaling and threshold selection, underscoring that model outputs should be interpreted as QC-dependent prioritization scores rather than calibrated probabilities or definitive biological classifications. Overall, SeqQC-Former offers a reproducible post-calling QC-aware prioritization framework for large-scale somatic SNV review and underscores the importance of explicitly modeling sequencing-quality information when interpreting structured cancer genomics datasets.

Humans↗

PubMed2003

DNA polymerase X of African swine fever virus: insertion fidelity on gapped DNA substrates and AP lyase activity support a role in base excision repair of viral DNA.

DNA polymerase X (pol X) from African swine fever virus (ASFV) is the smallest naturally ocurring DNA-directed DNA polymerase (174 amino acid residues) described so far. Previous biochemical analysis has shown that ASFV pol X is a highly distributive, monomeric enzyme, lacking a proofreading 3'-5' exonuclease. Also, ASFV pol X binds intermediates of the single-nucleotide base excision repair (BER) process, and is able to efficiently repair single-nucleotide gapped DNA. In this work, we perform an extensive kinetic analysis of single correct and incorrect nucleotide insertions by ASFV pol X using different DNA substrates: (i) a primer/template DNA; (ii) a 1nt gapped DNA; (iii) a 5'-phosphorylated 1nt gapped DNA. The results obtained indicate that ASFV pol X exhibits a general preference for insertion of purine deoxynucleotides, especially dGTP opposite template C. Moreover, ASFV pol X shows higher catalytic efficiencies when filling in gapped substrates, which are increased when a phosphate group is present at the 5'-margin of the gap. Interestingly, ASFV pol X misinserts nucleotides with frequencies from 10(-4) to 10(-5), and the insertion fidelity varies depending on the substrate, being more faithful on a phosphorylated 1nt gapped substrate. We have analyzed the capacity of ASFV pol X to act on intermediates of BER repair. Although no lyase activity could be detected on preincised 5'-deoxyribose phosphate termini, ASFV pol X has lyase activity on unincised abasic sites. Altogether, the results support a role for ASFV pol X in reparative BER of damaged viral DNA during ASFV infection.

African Swine Fever↗

PubMed2000

Automated identification of amino acid sequence variations in proteins by HPLC/microspray tandem mass spectrometry.

Amino acid sequence variations resulting from single-nucleotide polymorphisms (SNPs) were identified using a novel mass spectrometric method. This method obtains 99+% protein sequence coverage for human hemoglobin in a single LC-microspray tandem mass spectrometry (microLC-MS/MS) experiment. Tandem mass spectrometry data was analyzed using a modified version of the computer program SEQUEST to identify the sequence variations. Conditions of sample preparation, chromatographic separation, and data collection were optimized to correctly identify amino acid changes in six variants of human hemoglobin (Hb C, Hb E, Hb D-Los Angeles, Hb G-Philadelphia, Hb Hope, and Hb S). Hemoglobin proteins were isolated and purified, dehemed, (S)-carboxyami-domethylated, and then subjected to a combination proteolytic digestion to obtain a complex peptide mixture with multiple overlaps in sequence. Reversed-phase chromatographic separation of peptides was achieved on-line with MS utilizing a robust fritless microelectrospray interface. Tandem mass spectrometry was performed on an ion trap mass spectrometer using automated data-dependent MS/MS procedures. Tandem mass spectra were collected from the five most abundant ions in each scan using dynamic and isotopic exclusion to minimize redundancy. The spectra were analyzed by a version of the SEQUEST algorithm modified to identify amino acid substations resulting from SNPs.

Amino Acid Sequence↗

PubMed2003

Multipoint linkage-disequilibrium mapping narrows location interval and identifies mutation heterogeneity.

Single-nucleotide polymorphism (SNP) genotypes were recently examined in an 890-kb region flanking the human gene CYP2D6. Single-marker and haplotype-based analyses identified, with genomewide significance (P < 10-7), a 403-kb interval displaying strong linkage disequilibrium (LD) with predicted poor-metabolizer phenotype. However, the width of this interval makes the location of causal variants difficult: for example, the interval contains seven known or predicted genes in addition to CYP2D6. We have developed the Bayesian fine-mapping software coldmap, which, applied to these genotype data, yields a 95% location interval covering only 185 kb and establishes genomewide significance for a causal locus within the region. Strikingly, our interval correctly excludes four SNPs, which individually display association with genomewide significance, including the SNP showing strongest LD (P < 10-34). In addition, coldmap distinguishes homozygous cases for the major CYP2D6 mutation from those bearing minor mutations. We further investigate a selection of SNP subsets and find that previously reported methods lead to a 38% savings in SNPs at the cost of an increase of <20% in the width of the location interval.

Bayes Theorem↗

PubMed2001

Loss of DNA polymerase beta stacking interactions with templating purines, but not pyrimidines, alters catalytic efficiency and fidelity.

Structures of DNA polymerases bound with DNA reveal that the 5'-trajectory of the template strand is dramatically altered as it exits the polymerase active site. This distortion provides the polymerase access to the nascent base pair to interrogate proper Watson-Crick geometry. Upon binding a correct deoxynucleoside triphosphate, alpha-helix N of DNA polymerase beta is observed to form one face of the binding pocket for the new base pair. Asp-276 and Lys-280 stack with the bases of the incoming nucleotide and template, respectively. To determine the role of Lys-280, site-directed mutants were constructed at this position, and the proteins were expressed and purified, and their catalytic efficiency and fidelity were assessed. The catalytic efficiency for single-nucleotide gap filling with the glycine mutant (K280G) was strongly diminished relative to wild type for templating purines (>15-fold) due to a decreased binding affinity for the incoming nucleotide. In contrast, catalytic efficiency was hardly affected by glycine substitution for templating pyrimidines (<4-fold). The fidelity of the glycine mutant was identical to the wild type enzyme for misinsertion opposite a template thymidine, whereas the fidelity of misinsertion opposite a template guanine was modestly altered. The nature of the Lys-280 side-chain substitution for thymidine triphosphate insertion (templating adenine) indicates that Lys-280 "stabilizes" templating purines through van der Waals interactions.

Arginine↗

PubMed2004

Association of protein kinase C alpha (PRKCA) gene with multiple sclerosis in a UK population.

Twin, family and adoption studies suggest that susceptibility to multiple sclerosis is substantially mediated by genetic factors. Linkage to human chromosome 17q, homologous to a locus linked to experimental animal models of multiple sclerosis, has been widely replicated and the region likely to harbour a multiple sclerosis susceptibility gene has recently been refined to a 2.5 Mb region of 17q22-24. The candidate multiple sclerosis susceptibility gene, protein kinase C alpha (PRKCA), maps within this interval and association with 35 single-nucleotide polymorphism (SNP) markers, spanning the gene with a median spacing of 7.8 kb, was tested using a case-control approach. Single-marker genotype and estimated haplotype frequencies were compared in UK unrelated cases with multiple sclerosis (n = 184) and healthy controls (n = 340) in order to investigate association with susceptibility to disease. A haplotype of two SNPs mapping to the proximal region of the gene showed evidence for association with susceptibility (Bonferroni-corrected P value = 1.1 x 10(-5)). These findings suggest that further investigation of the PRKCA gene is warranted, particularly in cohorts with evidence of linkage to 17q22. Most of the SNPs investigated in this study were intronic and screening to identify disease-associated functional mutations is now required. Our results suggest that the promoter and proximal gene region should be not only included but prioritized in any screening strategy.

Adult↗

PubMed2000

A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension.

A rapid, high throughput readout for single-nucleotide polymorphism (SNP) analysis was developed employing single base chain extension and cytometric analysis of an array of fluorescent microspheres. An array of fluorescent microspheres was coupled with uniquely identifying sequences, termed complementary ZipCodes (cZipCodes), which allowed for multiplexing possibilities. For a given assay, querying a polymorphic base involved extending an oligonucleotide containing both a ZipCode and a SNP-specific sequence with a DNA polymerase and a pair of fluoresceinated dideoxynucleotides. To capture the reaction products for analysis, the ZipCode portion of the oligonucleotide was hybridized with its cZipCodes on the microsphere. Flow cytometry was used for microsphere decoding and SNP typing by detecting the fluorescein label captured on the microspheres. In addition to multiplexing capability, the ZipCode system allows multiple sets of SNPs to be analyzed by a limited set of cZipCode-attached microspheres. A standard set of non-cross reactive ZipCodes was established experimentally and the accuracy of the system was validated by comparison with genotypes determined by other technologies. From a total of 58 SNPs, 55 SNPs were successfully analyzed in the first pass using this assay format and all 181 genotypes across the 55 SNPs were correct. These data demonstrate that the microsphere-based single base chain extension (SBCE) method is a sensitive and reliable assay. It can be readily adapted to an automated, high-throughput genotyping system. [Primer sequences used in this study are available as online supplementary materials at www.genome.org.]

DNA, Complementary↗

PubMed2003

Multiplex minisequencing screen for common Southeast Asian and Indian beta-thalassemia mutations.

BACKGROUND: Beta-thalassemia is endemic to many regions in Southeast Asia and India, and <20 beta-globin gene mutations account for > or =90% of beta-thalassemia alleles in these places. We describe a multiplex minisequencing assay to detect these common mutations. METHODS: Gap-PCR was used to simultaneously amplify the beta-globin gene from genomic DNA and to detect the Delta619bp deletion mutation. Multiplex minisequencing was then performed on the amplified beta-globin fragment to detect an additional 15 common Southeast Asian and Indian beta-thalassemia mutations. Site-specific primers of different lengths were subjected to multiple rounds of annealing and single-nucleotide extension in the presence of thermostable DNA polymerase and the four dideoxynucleotides, each labeled with a different fluorophore. Minisequencing products were separated and detected by capillary electrophoresis, followed by automated genotyping. The optimized assay was subjected to a double-blind validation analysis of 89 beta-thalassemia and wild-type DNA samples of known genotype. RESULTS: Homozygous wild-type or mutant DNA samples produced electropherograms containing only a single colored peak for each mutation site, whereas samples heterozygous for a specific mutation displayed two different-colored peaks for that mutation site. Samples were automatically genotyped based on color and position of primer peaks in the electropherogram. In the double-blind validation analysis, all 89 DNA samples were genotyped correctly (100% assay specificity). CONCLUSIONS: The described semiautomated multiplex minisequencing assay can detect the most common Southeast Asian and Indian beta-thalassemia mutations, is amenable to high-throughput scale up, and may bring population-based screening of beta-thalassemia in endemic regions a step closer to implementation.

Asia, Southeastern↗

PubMed2003

Typing single-nucleotide polymorphisms using a gel-based sequencer: a new data analysis tool and suggestions for improved efficiency.

Single-nucleotide polymorphisms (SNPs) are increasingly used as genetic markers. Although a high number of SNP-genotyping techniques have been described, most techniques still have low throughput or require major investments. For laboratories that have access to an automated sequencer, a single-base extension (SBE) assay can be implemented using the ABI SNaPshot trade mark kit. Here we present a modified protocol comprising multiplex template generation, multiplex SBE reaction, and multiplex sample analysis on a gel-based sequencer such as the ABI 377. These sequencers run on a Macintosh platform, but on this platform the software available for analysis of data from the ABI 377 has limitations. First, analysis of the size standard included with the kit is not facilitated. Therefore a new size standard was designed. Second, using Genotyper (ABI), the analysis of the data is very tedious and time consuming. To enable automated batch analysis of 96 samples, with 10 SNPs each, we developed SNPtyper. This is a spreadsheet-based tool that uses the data from Genotyper and offers the user a convenient interface to set parameters required for correct allele calling. In conclusion, the method described will enable any lab having access to an ABI sequencer to genotype up to 1000 SNPs per day for a single experimenter, without investing in new equipment.

Computational Biology↗