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Low frequency DNA Variant discovery from Sanger Sequence Reads

Mutation detection has become increasingly important in the study of cancer. Mutation Surveyor identifies mutations from a physical comparison of sequence traces using our patented anti-correlation algorithm with the results shown in a mutation electropherogram. Compatible with all major sequencing platforms, Applied BioSystems’ Prism, Beckman Coulter CEQ as well as MegaBACE platforms the software automatically detects DNA variants, homozygous/heterozygous substitutions and duplications/deletions directly from sequence trace data through comparison of sample to a known reference trace using multiple factors to identify mutations. The anti-correlation method calculates the differences between the reference and sample traces, showing any physical variation in a mutation electropherogram. Traditional base calling software, such as the AB KB Basecaller, or other programs that compare base call text, (DNAStar, Sequencher, Variant Reporter) software are not capable of detecting the small percentage of Somatic mutations, heteroplasmy nor mosaicism within cells. The trace comparison method, however, does not rely on base calling and successfully identifies somatic mutations in cellular levels as low as 20 percent. Mutation Surveyor is beneficial in the study of cancer because the increased sensitivity of the software allows detection of somatic mutations often embedded within normal cells. Accuracy of the software in the bi-directional analysis mode is over 99%, with sensitivity to greater than 5% of the primary peak. The anti-correlation method also proves beneficial in the direct comparison of normal to cancer cells for the detection of somatic mutations. Somatic mutations occur at the rate of about 1-2ppm (parts per million) in cancer samples.

Low frequency peaks of the same color and same spatial position in both directions is detected by Mutation Surveyor, indicating a possible somatic mutation with a green bar in the Mutation Electropherogram.

Somatic mutation detection in tp53.  

The Somatic Variation detected in the illustration has a 6.15% mutant allele contribution as calculated by our Quantification tool. This also means that the software was able to pick up this low frequency variant even at 6% compared to the 93% normal population.

The software uses migration time changes of the sample versus the reference traces to locate homozygous and heterozygous indels. The software uses a log scale confidence score for each point mutation that involves four factors: signal to noise ratio, peak height, overlap, and intensity drop.

  • The signal to noise ratio is used to determine the confidence of the peaks.

  • The peak height is the highest peak intensity of the mutation peak in the electropherogram.

  • The overlapping factor is calculated by the reference peak and the sample peak of the different color. It is an indicator of the relative shift of the two peaks in the horizontal (time) direction.

  • The dropping factor indicates how much the vertical peak intensity has dropped relative to the neighboring peaks. The software uses 4 neighboring peaks to calculate the relative dropping factor.

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Mutation Surveyor Capabilities:
Detection Sensitivity & Accuracy
INDEL Detection
INDEL de-Convolution
Somatic Mutation Detection
Mutation Quantification
Methylation Analysis
Hypervariable Region Variant Detection
Reference Assembly
Custom Reporting Options
Alignment of Reads
Mitochondria DNA Sequence Analysis
Base Calling
Unattended Operation 

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