Reviewing VisCap Outputs

Overview

1. Start with the first page which represents all chromosomes

   • Solid light gray lines represent theoretical/ideal CNV thresholds
     • The top line represents 0.58 or where a true 1-copy gain would fall
     • The bottom line represents -1.00 or where a true 1-copy loss would fall
   • Solid black lines represent the LMM positive control fixed thresholds
     • The top line is positioned at 0.40
     • The bottom line is positioned at -0.55
   • Dashed lines represent the distribution of normal 2-copy exons
   • Exons that fall outside the dashed lines and the fixed thresholds are considered abnormal and are color coded as mentioned above [insert screenshot here]

2. Scan through all pages (chromosomes) for each patient. Each chromosome is further broken down by gene. [insert screenshot here]

Good Quality Gain/Loss VisCap Outputs

Full Chromosome View

1. The majority of data points cluster around “0” (copy number = 2)
2. The dotted lines are close together, indicating very little scattering of data points
3. There are a small number of called CNVs
4. Called CNVs are well separated from the adjacent points that do not pass the thresholds

Examples (L: Gain, R: Loss)

[insert screenshot here]

Individual Chromosome View

1. The majority of data points not passing the thresholds cluster around “0”
2. Called CNVs are well separated from the adjacent points that do not pass the thresholds
3. All consecutive exons within a called CNV have similar log2 ratios
4. The log2 ratios of called CNVs approach (or surpass) expected values for a 1-copy gain or loss [insert screenshot here]

Examples (L: Gain, R: Loss)

[insert screenshot here]

Anomalous VisCap Outputs

Example 1: Notice how there are “chunks” of exons that appear to be elevated or lowered with respect to the “0” normal line. Interestingly, these chunks represent the behavior of chromosomes or genes within the sample. This is likely sample specific and possibly a consequence of DNA extraction and purification. Some chromosomes may have trouble fully unfolding or uncondensing and therefore they are more difficult to access for extraction and subsequent assaying by molecular techniques such as Next Gen Sequencing and oligo capture or hybridization. Sample should probably be re-run for CNV analysis or reported as inconclusive. [insert screenshot here]

Example 2: The first page of the VisCap output shows a large number of losses. The log2 ratios for these calls vary widely and show no clustering around expected values for a true heterozygous or homozygous deletion. If the sample passes VisCap’s quality control filters, it will be necessary to proceed with the visual assessment; however, the sample quality should be categorized as “Pass – Scattered”. [insert screenshot here]

Example 3: Regions with poor mapping quality or problems with capture efficiency may exhibit patterns where log2 ratios gradually rise or fall. [insert screenshot here]

Identifying Failed Samples

VisCap performs an analysis on all samples in the batch. If any sample fails the program’s built-in quality control measures, the sample is automatically removed and the analysis is repeated on the remaining samples. VisCap will continue through this automatic process until all analyzed samples pass quality control. Each of these runs is stored as a separate folder, and the failed samples can be identified by reviewing the output files in each folder.