Why do I need controls?

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.

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Why positive controls are useful for your assay

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.

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What should I use as a positive control?

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:

  1. A positive sample from a study that has already been genotyped
  2. A DNA sample purchased from the NHGRI Sample Repository for Human Genetic Research at Coriell (Coriell Institute for Medical Research maintains an extensive repository of DNA for sale)
  3. A DNA fragment cloned into a plasmid
  4. A synthetic oligonucleotide

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Looking for human SNP positive controls in the sample repository at Coriell

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.

  1. Go to taqmanSNP and search for “C____490932_10”.
  2. A new page will load. Click “View Details”.
  3. In the Product Details section, click on the SNP ID (in this example “rs3204955”) which links out to a SNP page in the NCBI’s dbSNP database.
  4. Find the table titled “Allele” in the top header section (Figure 1). Look to see if there is MAF data from the 1000 Genomes Project available. If there is, go through the following steps to locate a sample in NCBI’s 1000 Genomes browser.
C____490932_10

Figure 1:  Allele table from NCBI’s dbSNP database

  1. Go to the 1000 Genomes browser at https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/
  2. Enter the SNP ID in the search field on the left hand side.
  3. A table containing multiple SNP IDs will populate the page but the ID you searched will be highlighted. 
  4. A list of Populations/Samples are listed on the left hand side of the page. 
  5. Click the expansion arrow on a population to see the individual sample identifiers.
  6. Choose a heterozygous or mutant allele homozygous sample (for example, here HG00103 is homozygous for the minor allele) and copy the sample ID (these begin with HG or NA).
  7. Go to the Coriell Institute for Medical Research website and paste the sample ID (beginning with HG or NA) into the search field and click search. If the Coriell DNA repository contains the sample, the item will come up. Select the DNA product for ordering.

Alternatively, go to the human Ensembl database and type in the SNP ID number (in this example “rs3204955”) in the upper right search bar.

  1. Click on “Sample Genotypes” from the left side menu.
  2. In the table titled “1000 Genomes Project Phase 3”, click “Show” directly next to the word “ALL” in the column labeled “Population".
  3. Choose a heterozygous or mutant allele homozygous sample (for example, here HG00103 is homozygous for the minor allele) and copy the sample ID.
  4. Go to the Coriell Institute for Medical Research website and paste the sample ID (beginning with HG or NA) into the search field and click search. If the Coriell DNA repository contains the sample, the item will come up. Select the DNA product for ordering.

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Centers for Disease Control and Prevention (CDC) reference materials

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.

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What if I can’t find a positive control cell line or gDNA?

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 techsupport@thermofisher.com to get help identifying an appropriate sequence that will encompass the amplicon.

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Plasmid controls

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.

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Oligonucleotide positive controls

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.

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References

1. Wang B, Steain M, Dwyer DE et al. (2011) Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: Drug resistance mutations in influenza virus as an example. Virol J 8:405.