An experimental control, such as a positive or negative control, provides a level of confidence in the data and are used to ensure the experiment was performed correctly. When a negative result is expected, the negative control should produce a negative result. Likewise, the positive control should produce a positive result. In a SNP genotyping experiment the negative control shows there is no contaminant in the reagents and that the fluorescence is not due to probe degradation. The positive control is not always necessary, but it is good laboratory practice to include at least one positive control containing the minor allele where the genotype is known for the SNP being interrogated, especially if the minor allele frequency (MAF) is low. The positive control aids in cluster calling for the analysis algorithms.
A laboratory may prefer to use samples that represent at least two genotype classes (homozygous major and minor, or homozygous major and heterozygous) to test that both assay probes function. Ideally, genomic DNA (gDNA) samples of known genotypes are used for such experiments.
To check for problems in your allelic discrimination (AD) plot, include a positive (or known) control as well as a no-template (negative) control. The negative control shows that a fluorescent signal in your samples is not likely due to probe degradation, and that there has been amplification in the samples by comparison. Positive control samples are not usually necessary with TaqMan SNP Genotyping Assays if both the sample population and the MAF is sufficiently large. However, if the MAF is relatively low, there may not be enough samples to detect that allele, making it difficult for the software to identify genotypes correctly. It makes sense to include a positive control if you are only running a few samples as this allows you to check that your assay set-up, sample preparation, instrument, and software are all functioning properly.
There are a number of sources of human positive control DNA sequences that are likely to include the SNP you are interrogating. Sources may include:
If a positive control is not readily available from a previous study, then you can check for a commercially available human gDNA sample from Coriell.
The following steps will take you from a TaqMan Assay on the Thermo Fisher Scientific website to ordering a control sample for that SNP (if one exists in the 1000 Genomes Project) from the Coriell repository.
Let's assume that you are looking for the SNP that is interrogated by assay C____490932_10.
Figure 1: Allele table from NCBI’s dbSNP database
Alternatively, go to the human Ensembl database and type in the SNP ID number (in this example “rs3204955”) in the upper right search bar.
The Centers for Disease Control and Prevention (CDC) provides genetic information on cell line DNAs that can be used as reference materials for genetic testing and assay validation for human samples. Some of these cell lines were characterized by the Genetic Testing Reference Materials Coordination Program (GeT-RM). One major focus category is the ′Genetic Inherited Disease & Pharmacogenetics′ section. You can download tables of reference samples that contain pharmacogenomics (PGx)/drug metabolism enzyme (DME) or disease allele variants, many of which have been confirmed by multiple labs and genetic testing technologies, from the CDC website.
If you can’t find the SNP sequence you are looking for in the Coriell repository, there are a number of companies, including Thermo Fisher Scientific, that will produce oligonucleotides or plasmids with your variant of interest. If the assay you use is a pre-designed assay you can contact technical support at firstname.lastname@example.org to get help identifying an appropriate sequence that will encompass the amplicon.
Genomic DNA (gDNA) and plasmids containing cloned target sequences are commonly used as standards in quantitative PCR. In addition to major or minor allele sequences encompassing assay amplicon sequences, we recommend that plasmids contain the RNase P RPPH1 gene to enable quantitation of plasmids using the TaqMan RNase P assay before use in genotyping experiments. This way, you can normalize the amount of plasmid by getting Ct values close to those of your samples. Contact technical support for more information.
Equal quantities of major and minor allele plasmids are mixed together to create heterozygous controls. Alternatively, heterozygous controls can be ordered that contain sequences for both alleles. Use the homozygous and heterozygous plasmid controls to demonstrate amplification and detection of all three genotypes by a given assay. Plasmid control samples may not always cluster with gDNA samples. This is likely caused by non-target sequences in gDNA contributing a small background that may shift the location of genotype clusters.
Oligonucleotide positive controls are another alternative to gDNA and plasmids. For example, researchers have produced oligonucleotide positive controls of influenza gene mutations for use in PCR—this would be of great importance, for instance, during a pandemic when genotyping of variants has to be fast, accurate, and reliable (1).
Note that plasmid and synthetic sequences are highly abundant, so they can be sources of PCR contamination in a lab. You should exercise great care when working with synthetic DNA controls to prevent their contamination of stock PCR reagents (solvents used for the dilution of PCR reagents and laboratory equipment and surfaces).
Identifying appropriate positive controls for your SNP genotyping assays is central to good assay setup and will make it easier to interpret results in the long run.
For Research Use Only. Not for use in diagnostic procedures.