Why controls matter
When assessing genotypes, especially those with very low allelic frequencies, you may not have gDNA samples representing all known genotypes. Problems assigning genotypes can also arise when the angle of separation between the clusters is not maximal. In such situations, it is critical to perform experiments with controls for each potential genotype. Without controls, genotyping software may be unable to associate (“call”) the samples with the correct genotype(s). Including a complete set of controls will greatly improve accuracy and confidence of both manual and automated genotype calling for your samples.
Simple genotyping controls
Genotyping controls can be generated as double-stranded DNA (dsDNA) fragments. IDT gBlocks Gene Fragments make this easy, and the rhAMP® SNP ordering tools make this convenient. When rhAmp SNP Assays are designed, both allelic sequences will be made available as gBlocks Gene Fragments for ordering (the heterozygous control is created by mixing the 2 homozygous sequences at equal ratio). gBlocks Gene Fragments are sequence-verified, dsDNA fragments available in both tube and plate formats, and ship in only a few working days. They are already frequently used as qPCR controls to generate standard curves for absolute quantification (for more details, see Easily designed standard curves for qPCR). Here, we show how they can be a useful tool for genotyping data analysis.
Figure 1 shows allelic discrimination plots for the detection of SNP rs72558187 in the Cytochrome P450 2C9 (CYP2C9) gene in human blood samples. All the samples in this experiment are homozygous for allele 1 (Homo 1). In Panel A, a single cluster is present, but without controls, the genotype of the samples cannot be appropriately called by the genotyping software.
In Panel B, 1000 copies of gBlocks Gene Fragments representing each genotype were run separately, in the same plate, and then included in the sample analysis. By including these controls, the software could correctly call the allele in the sample DNA, in this case, to Homo 1.
Figure 1. gBlocks Gene Fragment genotyping controls improve autocalling. Human gDNA (10 ng) samples were purified from whole blood (QIAamp® DNA blood kit, Qiagen) and genotyped for SNP rs72558187 in the Cytochrome P450 2C9 (CYP2C9) gene on a QuantStudio® 7 Flex Real-Time PCR System (Applied Biosystems). (A) No gBlocks controls were included in the analysis. The black x’s represent unassigned alleles. (B) The same experiment performed with the addition of positive controls (1000 copies of gBlocks controls for each allele), assigned accordingly: Homo1 = red, Het = blue, and Homo2 = green.
Tips for using gBlocks Gene Fragments as genotyping controls
- gBlocks fragment control sequences for the homozygous alleles are provided when an rhAmp SNP Genotyping assay is selected (or designed). Once obtained, create the heterozygous control by mixing the 2 homozygous sequences at equal ratio.
- For cost savings, when designing controls for multiple SNPs, amplicons can be combined onto a single gBlocks Gene Fragment. For example, an amplicon for the reference alleles (the base found in the reference genome) from distinct SNPs can be designed onto one gBlocks Gene Fragment, and those for the alternative alleles can be designed onto a second gBlocks Gene Fragment.
- We find that the best controls consist of 1000 copies gBlocks Gene Fragment representing each genotype (run separately, but in the same plate). Calculate copy number as follows:
(C) (M) (1 x 10–15 mol/fmol) (Avogadro’s number) = copy number/µL
Where C is the current concentration of the gBlocks Gene Fragment in ng/µL, and M is the molecular weight in fmol/ng, as provided on the spec sheet.
See the article, Tips for working with gBlocks Gene Fragments, for more details.
- Run the gBlocks Gene Fragments on the same plate with DNA samples.
Run at least 2 replicates of the gBlocks Gene Fragment controls when using automated genotype calling.
- Save time by using IDT predesigned gBlocks fragment genotyping control sequences. Download these sequences for ordering when selecting rhAmp SNP Genotyping assays (Figure 2).
Order gBlocks fragment controls when selecting genotyping assays
gBlocks Gene Fragments are useful in many applications, including as genotyping controls
gBlocks Gene Fragments enable easy gene construction or modification, and can be used in applications such as antibody research and CRISPR-mediated genome editing. While they have been used as qPCR standards, they are now finding value as standards in genotyping assays.
Select from millions of up-to-date, predesigned rhAmp Genotyping Assays
The IDT rhAmp predesigned assay library contains designs for >10 million human SNPs with minor allele frequencies >1%. They include a broad selection of functionally validated ADME SNP assays [i.e., assays that target SNPs in genes that are responsible for the absorption, distribution, metabolism, and excretion (ADME) of pharmaceutical compounds]. These rhAmp Genotyping Assays demonstrate consistently high performance with >99.5% call accuracy in over 90% assays tested. A tool for generating custom assay designs that uses the same bioinformatics approach as for the predesigned assays is also available for newly discovered human SNPs or assay designs for other species.
Download gBlocks Gene Fragments sequences already designed for each assay
As you order your rhAmp Genotyping Assays, you will have the opportunity to select gBlocks fragment controls for that assay at the same time. Figure 2 shows how to obtain the specific gBlocks Gene Fragment sequences of the alleles for a particular assay. Once you have identified the assay of interest by checking the box at the upper left-hand corner, simply click on the Download files menu, and you will be able to download the reference and alternate allele sequence amplicons as gBlocks Gene Fragments. These can then be ordered by visiting the gBlocks Gene Fragments product page.
Figure 2. Easy access to gBlocks fragment control sequences. Each predesigned rhAmp Genotyping Assay has associated gBlocks Gene Fragments sequences available to order for use as controls.
Ensure confidence in your genotyping data by using gBlocks Gene Fragments as control templates.