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Low frequency DNA Variant discovery from Sanger Sequencing Reads

Mutation Surveyor software's patented anti-correlation technology provides the enhanced sensitivity needed for the detection of low frequency alleles in Sanger sequencing traces, including low frequency variants associated with somatic mutations, heteroplasmy, and mosaicism. Many software packages such as DNAStar, Sequencher, and Variant Reporter rely on basecall differences and have been optimized for variant detection with alleles having heterozygous mixtures of 50%. However, today's sequencing projects demand software with an analytic sensitivity capable of detecting low frequency mutations that are often buried in background noise. Mutation Surveyor provides a solution to these issues by offering a unique trace to trace comparison of sample sequence traces to a reference trace, calculating and displaying the physical differences between sample and reference peak in a mutation electropherogram. This technology has demonstrated accuracy in the bi-directional analysis mode over 99%, with sensitivity to greater than 5% of the primary peak. The sensitivity of the anti-correlation method has proven to be beneficial in the direct comparison of normal to cancer cells for the detection of somatic mutations in Sanger sequencing traces from all major capillary electrophoresis outputs.

 

Mutation Surveyor Detects Minor Alleles Buried in the Baseline

 

 

Low frequency G>GA mutation detected in Mutation Surveyor. A green box in the Mutation Electropherogram indicates the presence of a possible low frequency variant.

 

Mutation Surveyor's trace to trace anti-correlation technology evaluates physical characteristics of the sample trace relative to the reference, including: signal to noise ratio, overlapping factor, and dropping factor. Often somatic/low frequency alleles do not result in major changes to the primary peak. However, Mutation Surveyor's sensitivity is capable of detecting these variants by monitoring minor mutation peak intensity calculated in the mutation electropherogram. When minor alleles of the same color are detected in both the forward and reverse direction, Mutation Surveyor places a green box at the location of event. The above image represents a low frequency peak detected in exon 5 of the tp53 gene.

 

Mutation Quantification Tool Provides Major and Minor Peak Contribution

 

 

The Mutation Quantifier report displays the contributions from each allele. The low frequency variant in this example had a minor/mutant peak contribution near 6% in both the forward and reverse trace.

 

The Mutation Quantification tool of the software automatically detects the major and minor allele contribution through two methods. Standardized allele ratio provides the percent decrease in normal intensity and the percent increase in mutant intensity and simplified allele ratio provides the relative percent contribution of the major and minor allele in Relative Fluorescence Units (RFU). The simplified allele ratio report indicates that the low frequency variants in this sample were detected with only 6% contribution of the primary peak.

 

 

Mutation Electropherogram window displaying the major and minor allele of the forward and reverse trace. This window can be accessed from the Mutation Quantifier report.

 

Flexible Reporting Options

 

 

The Custom Report lists possible low frequency mutations with a teal background. Custom color schemes can be added, and mutations can be edited with the click of a mouse.

 

The interactive reporting and display options of Mutation Surveyor provide the flexibility needed for the user to make edits to add, delete, or confirm low frequency mutations in a number of ways. Rapid customization of reporting options also provides useful ways to identify low frequency mutations, including background and text color coding. Users can easily add mutations to the Graphic Analysis Display at locations of possible somatic mutations and the mutation report will populate with the changes.

 

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Automated Somatic Mutation Detection in DNA Sequence Traces

 

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