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Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data.

ABSTRACT: Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotype calling based on the full course of real-time PCR data. Best cycle genotyping algorithm (BCGA), written in the open source language R, is based on the assumptions that classification depends on the time (cycle) of amplification and that it is possible to identify a best discriminating cycle for each SNP assay. The algorithm is unique in that it classifies samples according to the behavior of blanks (no DNA samples), which cluster with heterozygous samples. This method of classification eliminates the need for positive controls and permits accurate genotyping even in the absence of a genotype class, for example when one allele is rare. Here, we describe the algorithm and test its validity, compared to the standard end-point method and to DNA sequencing.

SUBMITTER: Callegaro A

PROVIDER: S-EPMC1440877 | biostudies-other | 2006

REPOSITORIES: biostudies-other

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Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data.

Callegaro A A Spinelli R R Beltrame L L Bicciato S S Caristina L L Censuales S S De Bellis G G Battaglia C C

Nucleic acids research 20060414 7

Single nucleotide polymorphisms (SNPs) are often determined using TaqMan real-time PCR assays (Applied Biosystems) and commercial software that assigns genotypes based on reporter probe signals at the end of amplification. Limitations to the large-scale application of this approach include the need for positive controls or operator intervention to set signal thresholds when one allele is rare. In the interest of optimizing real-time PCR genotyping, we developed an algorithm for automatic genotyp ...[more]

PMID: 16617143

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Project description:Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.

Project description:BACKGROUND:Polyploidy is widespread in animals and especially in plants. Different kinds of ploidies exist, for example, hexaploidy in wheat, octaploidy in strawberries, and diploidy, triploidy, tetraploidy, and pseudo-tetraploidy (partly tetraploid) in fish. Triploid offspring from diploid parents occur frequently in the wild in Atlantic salmon (Salmo salar) and, as with triploidy in general, the triploid individuals are sterile. Induced triploidy in Atlantic salmon is common practice to produce sterile fish. In Norwegian aquaculture, production of sterile triploid fish is an attempt by government and industry to limit genetic introgression between wild and farmed fish. However, triploid fish may have traits and properties that differ from those of diploids. Investigating the genetics behind traits in triploids has proved challenging because genotype calling of genetic markers in triploids is not supported by standard software. Our aim was to develop a method that can be used for genotype calling of genetic markers in triploid individuals. RESULTS:Allele signals were produced for 381 triploid Atlantic salmon offspring using a 56 K Thermo Fisher GeneTitan genotyping platform. Genotypes were successfully called by applying finite normal mixture models to the (transformed) allele signals. Subsets of markers were filtered by quality control statistics for use with downstream analyses. The quality of the called genotypes was sufficient to allow for assignment of diploid parents to the triploid offspring and to discriminate between maternal and paternal parents from autosomal inheritance patterns. In addition, as the maternal inheritance in triploid offspring is identical to gynogenetic inheritance, the maternal recombination pattern for each chromosome could be mapped by using a similar approach as that used in gene-centromere mapping. CONCLUSIONS:We show that calling of dense marker genotypes for triploid individuals is feasible. The resulting genotypes can be used in parentage assignment of triploid offspring to diploid parents, to discriminate between maternal and paternal parents using autosomal inheritance patterns, and to map the maternal recombination pattern using an approach similar to gene-centromere mapping. Genotyping of triploid individuals is important both for selective breeding programs and unravelling the underlying genetics of phenotypes recorded in triploids. In principle, the developed method can be used for genotype calling of other polyploid organisms.

Dataset Information

Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data.

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Algorithm for automatic genotype calling of single nucleotide polymorphisms using the full course of TaqMan real-time data.

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