> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/microarray/general/microarray-general-troubleshooting-list/000002130.md).
# How to check Infinium samples for possible cross-sample contamination in GenomeStudio
### **How Copy Number Relates to Sample Contamination**
The Illumina Genome Viewer (IGV) tool in GenomeStudio 2.0 can be used to determine the number of different genotypes present in a sample. This is the basis of Copy Number Variation (CNV) analysis, but it can also be used to detect cross-sample contamination. For diploid organisms, including humans, the expectation is to have three genotypes (AA, AB, BB) for autosomal chromosomes; the frequencies of the B-alleles are 0, 0.5, and 1, respectively. If DNA is present from multiple individuals, intermediate B-Allele Frequencies (BAF) are observed (eg, AAAB has a BAF of 0.25, and ABBB has a BAF of 0.75). If the intermediate BAF is observed to occur across the entire genome, this is likely caused by cross-sample contamination rather than copy number variation.
When Infinium data do not meet Call Rate specifications, it can be helpful for troubleshooting to determine if the failures are due to cross-sample contamination vs. noisy data (caused by processing issues and/or low quality/degraded input DNA).
**How to Check Samples for Cross-Sample Contamination in GenomeStudio**
1. In the GenomeStudio project, select **Tools** > **Show Genome Viewer.**
2. Select sample(s) by marking the check box next to the Sample name in **Table-Sample-SubColumn.**
3. In the **SubColumn** section, scroll down and check the boxes for **B Allele Freq** and **Log R Ratio.**
4. Select **Add to favorite** then **OK.**
**Figure 1.** Selecting Samples and SubColumns in the IGV Data Workspace
**Note:** To change the samples selected, under the **IGV Data Workspace** > **Data Plots** tab, select the leftmost Add icon (two squares and a + icon) and update **Sample** or **Subcolumn** selection.
**Figure 2.** Changing the samples selected
5. Select the **Update** button to display the B-Allele Freq (BAF) and Log R Ratio (LRR) plots.\
Select the **Chromosome Browser** by navigating **View** > **Chromosome Browser.** The Chromosome Browser tab shows the plots of multiple samples simultaneously; whereas, the Genome Viewer tab displays only one sample at a time.
**Figure 3.** Selection to display the B-Allele Frequency and Log R Ratio Plots
6. By default, the Chromosome Browser is zoomed in to display the region of the chromosome indicated by the red box on the schematic of the chromosome below the B-Allele Freq and Log R Ratio plots. It is often easier to notice patterns if more data is viewed. Adjust the zoom to view an entire chromosome by selecting the **'Zoom to Chromosome'** icon in the tool bar.
**Figure 4.** View of the B-Allele Frequency and Log R Ratio Plots in the Chromosome Browser
7. Non-contaminated samples have three lines on the B-Allele Frequency plot, running at 0, 0.5, and 1 (representing AA, AB, and BB genotypes respectively). The Log R Ratio plot is, ideally, a narrow, straight line running at 0. Contaminated samples have more than the expected three bands in the BAF plot.
### **Examples of B-Allele Frequency and Log R Ratio Plots from different data types**
#### **Clean Data:**
* BAF: Three lines running at 0, 0.5, 1 (Exceptions: X Chromosome in Males, Y Chromosome in Females).
* LRR is one line, running in a narrow band centered at y = 0.
**Figure 5.** B-Allele Frequency and Log R Ratio Plots of a Clean Data
***
####
#### **Noisy Data:**
* BAF: No clear distinct banding pattern of genotype nodes (regular pattern is barely detectable beneath the noise).
* LRR: A very thick band with scattered 'waterfall' of data points.
**Figure 6.** B-Allele Frequency and Log R Ratio Plots of a Noisy Data
***
####
#### **Cross-sample Contaminated Data:**
* BAF: Extra nodes present (red arrows). The number of chromosomes present is the total number of nodes -1. In this example, there are a total of seven nodes, meaning a total of six chromosomes are present. Since each sample contributes two chromosomes, a total of three samples are present.
* LRR: One clean line running at 0.
* **Note:** it is possible for data to be both noisy and contaminated.
**Figure 7.** B-Allele Frequency and Log R Ratio Plots of a Cross-sample Contaminated Data
***
#### **Figure 8.** Additional Examples of B-Allele Frequency Plots from different samples
Abnormal B-Allele Frequency plots can also be indicative of Copy Number Variations (CNV) (Figure 8, Number 3 above). CNV regions can be distinguished from cross-sample contamination by checking whether the B-Allele Frequency nodes are present across all chromosomes (cross-sample contamination) or only in certain regions of the genome (possible CNV). True CNV regions are also reflected in the LRR plot as non-zero regions.
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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2130), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000002130%20-%20Microarray%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |
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