Population genetic analysis of Plasmodium falciparum parasites using a customized Illumina GoldenGate genotyping assay

The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (n = 306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum.     

High-throughput SNP genotyping with the GoldenGate assay in maize

Single nucleotide polymorphisms (SNPs) are abundant and evenly distributed throughout the genomes of most plant species. They have become an ideal marker system for genetic research in many crops. Several high throughput platforms have been developed that allow rapid and simultaneous genotyping of up to a million SNP markers. In this study, a custom GoldenGate assay containing 1,536 SNPs was developed based on public SNP information for maize and used to genotype two recombinant inbred line (RIL) populations (Zong3 x 87-1, and B73 x By804) and a panel of 154 diverse inbred lines. Over 90% of the SNPs were successfully scored in the diversity panel and the two RIL populations, with a genotyping error rate of less than 2%. A total of 975 SNP markers detected polymorphism in at least one of the two mapping populations, with a polymorphic rate of 38.5% in Zong3 x 87-1 and 52.6% in B73 x By804. The polymorphic SNPs in B73 x By804 have been integrated with previously mapped simple sequence repeat markers to construct a high-density linkage map containing 662 markers with a total length of 1,673.7 cM and an average of 2.53 cM between two markers. The minor allelic frequency (MAF) was distributed evenly across 10 continued classes from 0.05 to 0.5, and about 16% of the SNP markers had a MAF below 10% in the diversity panel. Polymorphism rates for individual SNP markers in pair-wise comparisons of genotypes tested ranged from 0.3 to 63.8% with an average of 36.3%. Most SNPs used in this GoldenGate assay appear to be equally useful for diversity analysis, marker-trait association studies, and marker-aided breeding.     

High-throughput genotyping with the GoldenGate assay in the complex genome of soybean

Large numbers of single nucleotide polymorphism (SNP) markers are now available for a number of crop species. However, the high-throughput methods for multiplexing SNP assays are untested in complex genomes, such as soybean, that have a high proportion of paralogous genes. The Illumina GoldenGate assay is capable of multiplexing from 96 to 1,536 SNPs in a single reaction over a 3-day period. We tested the GoldenGate assay in soybean to determine the success rate of converting verified SNPs into working assays. A custom 384-SNP GoldenGate assay was designed using SNPs that had been discovered through the resequencing of five diverse accessions that are the parents of three recombinant inbred line (RIL) mapping populations. The 384 SNPs that were selected for this custom assay were predicted to segregate in one or more of the RIL mapping populations. Allelic data were successfully generated for 89% of the SNP loci (342 of the 384) when it was used in the three RIL mapping populations, indicating that the complex nature of the soybean genome had little impact on conversion of the discovered SNPs into usable assays. In addition, 80% of the 342 mapped SNPs had a minor allele frequency >10% when this assay was used on a diverse sample of Asian landrace germplasm accessions. The high success rate of the GoldenGate assay makes this a useful technique for quickly creating high density genetic maps in species where SNP markers are rapidly becoming available. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply approval of a product to the exclusion of others that may be suitable.     

Single nucleotide polymorphism genotyping using allele-specific PCR and fluorescence melting curves

We present a PCR method for identification of single nucleotide polymorphisms (SNPs), using allele-specific primers designed for selective amplification of each allele. Matching the SNP at the 3' end of the forward or reverse primer, and additionally incorporating a 3' mismatch to prevent amplification of the incorrect allele, results in selectivity of the allele-specific primers. DNA melting analysis with fluorescent SYBR Green affords detection of the PCR products. By incorporating a GC-rich sequence into one of the two allele-specific primers to increase the melting temperature, both alleles can be measured simultaneously at their respective melting temperatures. Applying the DNA melting analysis to SNPs in ApoE and ABCA1 yielded results identical to those obtained with other genotyping methods. This provides a cost-effective, high-throughput method for amplification and scoring of SNPs.     

Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA

Single nucleotide polymorphism (SNP) is informative for human identification, and much shorter regions are targeted in analysis of biallelic SNP compared with highly polymorphic short tandem repeat (STR). Therefore, SNP genotyping is expected to be more sensitive than STR genotyping of degraded human DNA. To achieve simple, economical, and sensitive SNP genotyping for identification of degraded human DNA, we developed 18 loci for a SNP genotyping technique based on the mini-primer allele-specific amplification (ASA) combined with universal reporter primers (URP). The URP/ASA-based genotyping consisted of two amplifications followed by detection using capillary electrophoresis. The sizes of the target genome fragments ranged from 40 to 67 bp in length. In the Japanese population, the frequencies of minor alleles of 18 SNPs ranged from 0.36 to 0.50, and these SNPs are informative for identification. The success rate of SNP genotyping was much higher than that of STR genotyping of artificially degraded DNA. Moreover, we applied this genotyping method to case samples and showed successful SNP genotyping of severely degraded DNA from a 4-year buffered formalin-fixed tissue sample for human identification.     

Sequence polymorphism in polyploid wheat and their d-genome diploid ancestor

Sequencing was used to investigate the origin of the D genome of the allopolyploid species Triticum aestivum and Aegilops cylindrica. A 247-bp region of the wheat D-genome Xwye838 locus, encoding ADP-glucopyrophosphorylase, and a 326-bp region of the wheat D-genome Gss locus, encoding granule-bound starch synthase, were sequenced in a total 564 lines of hexaploid wheat (T. aestivum, genome AABBDD) involving all its subspecies and 203 lines of Aegilops tauschii, the diploid source of the wheat D genome. In Ae. tauschii, two SNP variants were detected at the Xwye838 locus and 11 haplotypes at the Gss locus. Two haplotypes with contrasting frequencies were found at each locus in wheat. Both wheat Xwye838 variants, but only one of the Gss haplotypes seen in wheat, were found among the Ae. tauschii lines. The other wheat Gss haplotype was not found in either Ae. tauschii or 70 lines of tetraploid Ae. cylindrica (genomes CCDD), which is known to hybridize with wheat. It is concluded that both T. aestivum and Ae. cylindrica originated recurrently, with at least two genetically distinct progenitors contributing to the formation of the D genome in both species.     

Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping

High-resolution melting of polymerase chain reaction (PCR) products can detect heterozygous mutations and most homozygous mutations without electrophoretic or chromatographic separations. However, some homozygous single nucleotide polymorphism (SNPs) have melting curves identical to that of the wild-type, as predicted by nearest neighbor thermodynamic models. In these cases, if DNA of a known reference genotype is added to each unknown before PCR, quantitative heteroduplex analysis can differentiate heterozygous, homozygous, and wild-type genotypes if the fraction of reference DNA is chosen carefully. Theoretical calculations suggest that melting curve separation is proportional to heteroduplex content difference and that the addition of reference homozygous DNA at one seventh of total DNA results in the best discrimination between the three genotypes of biallelic SNPs. This theory was verified experimentally by quantitative analysis of both high-resolution melting and temperature-gradient capillary electrophoresis data. Reference genotype proportions other than one seventh of total DNA were suboptimal and failed to distinguish some genotypes. Optimal mixing before PCR followed by high-resolution melting analysis permits genotyping of all SNPs with a single closed-tube analysis.     

Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9

A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC-ESI-MS a potent tool for the routine characterization of SNPs in forensic science.     

Single nucleotide polymorphism discovery in common bean

Single nucleotide polymorphisms (SNPs) were discovered in common bean (Phaseolus vulgaris L.) via resequencing of sequence-tagged sites (STSs) developed by PCR primers previously designed to soybean shotgun and bacterial artificial chromosome (BAC) end sequences, and by primers designed to common bean genes and microsatellite flanking regions. DNA fragments harboring SNPs were identified in single amplicons from six contrasting P. vulgaris genotypes of the Andean (Jalo EEP 558, G 19833, and AND 277) and Mesoamerican (BAT 93, DOR 364, and Rudá) gene pools. These genotypes are the parents of three common bean recombinant inbred line mapping populations. From an initial set of 1,880 PCR primer pairs tested, 265 robust STSs were obtained, which could be sequenced in each one of the six common bean genotypes. In the resulting 131,120?bp of aligned sequence, a total of 677 SNPs were identified, including 555 single-base changes (295 transitions and 260 transversions) and 122 small nucleotide insertions/deletions (indels). The frequency of SNPs was 5.16 SNPs/kb and the mean nucleotide diversity, expressed as Halushka??s theta, was 0.00226. This work represents one of the first efforts aimed at detecting SNPs in P. vulgaris. The SNPs identified should be an important resource for common bean geneticists and breeders for quantitative trait locus discovery, marker-assisted selection, and map-based cloning. These SNPS will be also useful for diversity analysis and microsynteny studies among legume species.     

Single nucleotide polymorphism in wheat chromosome region harboring Fhb1 for Fusarium head blight resistance

Fusarium head blight (FHB) is a destructive disease that reduces wheat grain yield and quality. To date, the quantitative trait locus on 3BS (Fhb1) from Sumai 3 has shown the largest effect on FHB resistance. Single nucleotide polymorphism (SNP) is the most common form of genetic variation and is suitable for high-throughput marker-assisted selection (MAS). We analyzed SNPs derived from 23 wheat expressed sequence tags (ESTs) that previously mapped near Fhb1 on chromosome 3BS. Using 71 Ning 7840/Clark BC7F7 recombinant inbred lines and the single-base extension method, we mapped seven SNP markers between Xgwm533 and Xgwm493, flanking markers for Fhb1. Five of the SNPs explained 45–54% of the phenotypic variation for FHB resistance. Haplotype analysis of 63 wheat accessions from eight countries based on SNPs in EST sequences, simple sequence repeats, and sequence tagged sites in the Fhb1 region identified four major groups: (1) US-Clark, (2) Asian, (3) US-Ernie, and (4) Chinese Spring. The Asian group consisted of Chinese and Japanese accessions that carry Fhb1 and could be differentiated from other groups by marker Xsnp3BS-11. All Sumai 3-related accessions formed a subgroup within the Asian group and could be sorted out by Xsnp3BS-8. The SNP markers identified in this study should be useful for MAS of Fhb1 and fine mapping to facilitate cloning of the Fhb1 resistance gene.     

A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E

We describe a new technique by which single base changes in human genes can be conveniently detected. In this method the DNA fragment of interest is first amplified using the polymerase chain reaction with an oligonucleotide primer biotinylated at its 5'-end. The amplified 5'-biotinylated DNA is immobilized on an avidin matrix and rendered single-stranded. The variable nucleotide in the immobilized DNA is identified by a one-step primer extension reaction directed by a detection step primer, which anneals to the DNA immediately upstream of the site of variation. In this reaction a single labeled nucleoside triphosphate complementary to the nucleotide at the variable site is incorporated. The method is highly sensitive, allowing the use of nucleoside triphosphates labeled with radioisotopes of low specific activity (3H) as well as nonradioactive markers (digoxigenin). The procedure consists of few and simple operations and is thus applicable to the analysis of large numbers of samples. Here we applied it to the analysis of the three-allelic polymorphism of the human apolipoprotein E gene. We were able to correctly identify all possible combinations of the three apo E alleles.     

Bayesian estimation of genomic copy number with single nucleotide polymorphism genotyping arrays

Background

The identification of copy number aberration in the human genome is an important area in cancer research. We develop a model for determining genomic copy numbers using high-density single nucleotide polymorphism genotyping microarrays. The method is based on a Bayesian spatial normal mixture model with an unknown number of components corresponding to true copy numbers. A reversible jump Markov chain Monte Carlo algorithm is used to implement the model and perform posterior inference.

Results

The performance of the algorithm is examined on both simulated and real cancer data, and it is compared with the popular CNAG algorithm for copy number detection.

Conclusions

We demonstrate that our Bayesian mixture model performs at least as well as the hidden Markov model based CNAG algorithm and in certain cases does better. One of the added advantages of our method is the flexibility of modeling normal cell contamination in tumor samples.     

Single nucleotide polymorphism for animal fibre identification

Animal fibres are highly valuable industrial products often adulterated during marketing. Currently, there is no precise method available to identify and differentiate the fibres. In this study, a PCR-RFLP technique was exploited to differentiate cashmere and wool fibres derived from goat and sheep, respectively. The presence of DNA in animal hair shafts has enabled the isolation of DNA from scoured cashmere and wool fibres. The mitochondrial cytochrome b sequences of both species were amplified by PCR using primers designed from conserved regions. The polymorphism observed between the two species was detected by restricting the amplified product by endonucleases viz., BamH1 and Ssp1. The RFLP profile clearly distinguishes the cashmere and wool fibres and this technique can also be exploited to test adulteration in animal fibres qualitatively.     

Single nucleotide polymorphism seeking long term association with complex disease

Successful investigation of common diseases requires advances in our understanding of the organization of the genome. Linkage disequilibrium provides a theoretical basis for performing candidate gene or whole-genome association studies to analyze complex disease. However, to constructively interrogate SNPs for these studies, technologies with sufficient throughput and sensitivity are required. A plethora of suitable and reliable methods have been developed, each of which has its own unique advantage. The characteristics of the most promising genotyping and polymorphism scanning technologies are presented. These technologies are examined both in the context of complex disease investigation and in their capacity to face the unique physical and molecular challenges (allele amplification, loss of heterozygosity and stromal contamination) of solid tumor research.     

Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay

Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of β-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the β-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.     

Multiplex single nucleotide polymorphism genotyping by adapter ligation-mediated allele-specific amplification

An improved approach for increasing the multiplex level of single nucleotide polymorphism (SNP) typing by adapter ligation-mediated allele-specific amplification (ALM-ASA) has been developed. Based on an adapter ligation, each reaction requires n allele-specific primers plus an adapter-specific primer that is common for all SNPs. Thus, only n+1 primers are used for an n-plex PCR amplification. The specificity of ALM-ASA was increased by a special design of the adapter structure and PCR suppression. Given that the genetic polymorphisms in the liver enzyme cytochrome P450 CYP2D6 (debrisoquine 4-hydroxylase) have profound effects on responses of individuals to a particular drug, we selected 17 SNPs in the CYP2D6 gene as an example for the multiplex SNP typing. Without extensive optimization, we successfully typed 17-plex SNPs in the CYP2D6 gene by ALM-ASA. The results for genotyping 70 different genome samples by the 17-plex ALM-ASA were completely consistent with those obtained by both Sanger's sequencing and PCR restriction fragment length polymorphism (PCR-RFLP) analysis. ALM-ASA is a potential method for SNP typing at an ultra-low cost because of a high multiplex level and a simple optimization step for PCR. High-throughput SNP typing could be readily realized by coupling ALM-ASA with a well-developed automation device for sample processing.