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Mutation Quantification in Sanger Sequence Traces

Quantification of allele ratios in gene expression and genomic analyses is useful for disease diagnosis and developing treatment protocols for patients in individualized medicine. For this reason we have included a unique functionality in Mutation Surveyor software to assist with the quantification of DNA variants discovered in Sanger Sequence reads generated by all major capillary sequencing platforms including Applied BioSystems Genetic Analyzers, Beckman Coulter CEQ as well as MegaBACE systems. Mutation Surveyor software physical anticorrelation technology, not present in other software programs such as Variant Reporter, DNAStar or Sequencher, quantifies DNA variants with an accuracy of +-10%.

Understanding the early stages of disease is invaluable since early detection is vital for efficacious treatment. Quantification of the presence of disease-causing markers can be used as a tool for early screening for diseases such as methylation status of genes associated with various cancers and heteroplasmy in mitochondrial DNA. Quantification of variant alleles can be used to monitor specific genes for drug-resistant mutations: BRC- ABL gene in chronic myelogenous leukemia (CML) patients and antiviral-target genes of hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Mutation quantification can be used to measure antibioticresistant mutations in bacterial strains within a sample, useful for monitoring infections and microbial source tracking.

Figure 1: Somatic Mutation Detection

Determining the concentration of different alleles in DNA can be problematic. Two alleles of one gene may differ at only one position with sequence identity for all other positions of the gene. Mutation detection within gene of cancer cells isolated from tumors may contain normal genes from co-isolated normal cells, resulting in a low frequency mutation allele. Promoter regions may be differentially or hyper-methylated and mtDNA may contain regions of heteroplasmy. Quantification of regions containing a high degree of variability may result in some positions not analyzed. Quantification of alleles that occur at low frequency may be below the detection limit of the technique used. The “Mutation Quantifier” function of Mutation Surveyor software solves the above problems. Mutation Surveyor software will align reference and sample sequences, detect mutations and quantify the wild type and variant alleles. Other methods used to determine such allelic variation may be just as robust but can be more expensive such as mass spectrometry TaqMan assay, and 2nd generation sequencing using Illumina/Solexa 1G Genomic Analyzer or Roche/454 FLX Genome Sequencers.

The “Mutation Quantifier” function will quantify the presence of multiple alleles at a specific location in the genome. Sanger sequencing traces are aligned to a reference sequence and mutations are identified (figure 1). Selecting the Mutation Quantifier function provides the quantity of the normal allele that is decreased at a specific locus in the sample and the quantity of the mutant allele that is gained at that position.

Standardized Allele Ratio

Figure 2

Quantification-Electropherogram window displaying three traces after clicking a cell in the DNA Quantification table. The gain of intensity for T in the sample is 67.38%(I). A decrease of intensity for C in the sample trace could not be calculated because all C positions are heterozygous. The position of interest is marked with a red dot in all electropherograms. The software uses the intensity ratio of current peak compared to the neighboring peaks of the same color in the same trace; short, red horizontal bars indicate Ts used for ratio calculation. Neighboring peaks at +1 and -1 bp will not be used to calculate the ratios because the intensity is often affected by the mutation. Single color peaks that may contain mosaic or heterozygous mutations will not be used to calculate ratios. Std1 (Standard 1—wild type or 0% mutation sample), Sample data file, Std2 (Standard 2—sample containing 100% or 50% mutation).

Simplified Allele Ratio

The Simplified Allele Ratio does not require any standards in order to quantify mutations. Instead, it looks directly at the trace peak to compare it to the reference. If the software detects a difference in wavelength, it will calculate a ratio of wildtype wavelength and the mutant wavelength within the peak.

Figure 3

The Simplified Allele Ratio calculated a 6.05% mutant and 93.95% normal for the peak. This is a possible somatic mutation detected in the TP53 gene by Mutation Surveyor.

Figure 3: Quantification Report

Both Standardized and Simplified Allele Ratio uses the same report. The report can list positions that contain mutation calls, positions of possible somatic mutations or specified positions of interests as selected in the settings. The column "Norm_percent" shows the quantification for the wildtype allele and the "Mut_Percent" shows the quantification for the mutant allele. By double clicking on any of the cells in the report, the user will be taken to that specific position as shown in Figure 2. This report can be saved and printed.

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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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