Mutation Surveyor, available in two batch processing capacities (400 & 48 traces per batch), uses patented anti-correlation technology to provide highly accurate discovery of DNA Variants from Sanger Sequencing traces. Compatible with outputs from Applied Biosystems, Beckman-Coulter and MegaBace systems, Mutation Surveyor rapidly locates all variants and SNPs & Indels between reference traces and sample/patient traces with excellent accuracy and sensitivity. The program is biologist-friendly, runs on Windows® Operating Systems, and has a myriad of reporting capabilities for both research and diagnostics applications.
Mutation Surveyor Capabilities:
Detection Sensitivity to 5%; Accuracy greater than 99%
Single as well as dual directional sequence traces are easily analyzed for any potential variants using our patented anti-correlation technology which performs an actual physical comparison of the sample/patient traces to the reference traces. Found differences are indicated by the sharp peaks in the mutation electropherogram.
Analysis courtesy of Dr. Yidong Bai, Assistant Professor, University of Texas Health Center, San Antonio TX
click
to enlarge
Accuracy of the software in the bi-directional analysis mode is over 99%,
with sensitivity to greater than 5% of the primary peak. |
Researchers no longer need to perform time consuming and inaccurate comparison of the entire sample trace. With Mutation Surveyor, any found variants of the sample, when compared to the wild type, are clearly indicated in our mutation electropherogram as a sharp peak.
Accuracy of the software in the bi-directional analysis mode is over 99%, with sensitivity to greater than 5% of the primary peak. Our collaborators have demonstrated an accuracy of 95% when processing single direction sequence traces.
Mutation Surveyor's detection sensitivity has been enhanced to report similar peaks that are buried in baseline noise of both the forward and reverse traces, alerting researchers to the possible presence of mutations buried in the background noise.
Mutation Surveyor easily processes 400 lanes of data in approximately two minutes, and can be operated on a fully automated unattended basis.
The software automatically forms contigs, performs alignments and mutation detection comparing both forward and reverse compliment patient traces to reference or normal traces. Homozygote and Heterozygote mutations are indicated by sharp peaks in our exclusive mutation electropherogram.
Unique algorithms perform a comparison of sample to reference traces in both forward and reverse electropherograms checking for similar peaks that are present in the background. This technique provides almost analytical quality sensitivity.
click
to enlarge
Homozygote Detection
Homozygote Variants are detected when the color of the reference drops 100% being replaced in the same spatial position by a second color in the sample or patient. |
|
click
to enlarge
Heterozygote Detection
The software detects heterozygote variants by observing a 50% drop in color of reference, with a second wavelength growing in approximately the same proportion and in the same spatial position.
|
Enhanced INDEL detection
Insertions and deletions are found by monitoring the mobility of the sample DNA fragments, again in comparison to the reference, providing an extremely low false positive rate and ignoring the miscalls or overcalls from basecaller software. In the event that a heterozygote insertion or deletion is detected, the software will de-convolute the multiple patient trace into two clean traces, while continuing with the mutation detection.
Het INDEL deconvolution
Download application note
Mutation Surveyor automatically deconvolutes sample traces when a Heterozygous Indel has occurred, shown in the figure. A is the reference trace, and B is the sample trace. The two portions of the convoluted sample trace, the Conserved Sector, C, and the Mutation Sector, D, can be separated into two clean traces following the point of the insertion or deletion. Then the software is able to shift the Mutation Sector D to align with the reference A. The software accurately detects the 19 deleted bases, shown in E and F.
A user may add a second heterozygous Indel in addition to the one the software automatically detected. The user may modify the start position, and the software will do the calculation based on user constraints.
Somatic Mutation Detection
Download application note
Mutation detection has become increasingly important in the study of cancer. Mutation Surveyor identifies mutations from a physical trace comparison using an anti-correlation algorithm with the results shown in a mutation electropherogram. The software automatically detects DNA variants 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 is not capable of detecting the small percentage of somatic mutations 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.
Somatic mutation detection in tp53.
 |
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.
|
Methylation Detection from Sanger Sequencing
Download application note
Methylation of DNA at the C of CpG sites has been associated with X chromosome inactivation, genomic imprinting, embryonic development, human diseases and differential gene expression. Epigenetic mechanisms involving methylation of DNA have been reported in colon cancer. Differential DNA methylation can be used to identify fetal DNA in maternal blood (4). Bisulfite treatment of DNA followed by sequencing is one method to analyze methylation of DNA. Improving speed and efficiency of sequence trace analysis will further the understanding of DNA methylation and its relationship to biological processes.
The GenBank sequence of the DNA of interest is used as a “ruler” to report nucleotide changes, including methylations and mutations. The methylation function of Mutation Surveyor utilizes the GenBank sequence text or derivatives of the sequence text and converts it into a synthetic reference sequence. This synthetic reference sequence is then physically compared to the sample sequence traces from bisulfite-treated DNA to find nucleotide differences.
Peak-by-peak comparison of the derivative and sample traces can be viewed in the Graphic Analysis Display window. Possible methylation sites will be identified in the mutation electropherogram:
 |
The top panel is the reference trace synthesized from the derivative GenBank file. The blue lines above the text indicate that the GenBank text was C (blue color). The second panel is the sample trace. The third panel is the mutation detection panel.
|
Mutation Quantification
Download application note
Quantification of allele ratios in gene expression and genomic analyses is useful for disease diagnosis and developing treatment protocols for patients in individualized medicine. 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 CML patients and antiviral-target genes of hepatitis C virus (HCV) and human immunodeficiency virus (HIV). Mutation quantification can be used to measure antibiotic-resistant mutations in bacterial strains within a sample, useful for monitoring infections and microbial source tracking.
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 from 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.
 |
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 [?%(D)]—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).
|
Hypervariable Region Analysis
Download application note
Organisms contain areas in the genome with hypervariability. A bacteriophage that infects Bordetella bronchiseptica, a relative of the bacterium that causes whooping cough, contains genes to specifically change its proteins, thus permitting binding to different cell receptors. These genes allow the phage to rapidly evolve new variants that can recognize and attack bacteria that may have become resistant to the previous phage. Similarly, infection by the influenza virus requires receptor binding through cell specific tropism (2). Several strains of Helicobacter pylori, the organism responsible for many cases of peptic ulcers and stomach cancer, show antibiotic resistance possibly due to efficiency of DNA repair or DNA polymerase (3). Additional causes for high mutation frequency come from antigenic variation found in sexually transmitted HIV (4) and the development of drug resistance mutations as in the case of the blood borne pathogen Hepatitis C virus (HCV).
Projects involving a high level of variability demand fully automated calls of SNPs and homozygous and heterozygous indels to identify variance between strains. Mutation Surveyor is the only software package that detects heterozygous indels by deconvoluting DNA sequence traces. Mutation Surveyor also provides an accuracy >99% in detecting homozygous and heterozygous SNPs when both forward and reverse traces are of Phred 20 quality. Mutation Surveyor features ease-of-use, low-learning curve, full automation, and an exclusive migration time comparison to detect Indels.
 |
 |
HCV sample (middle) shows high frequency of mutations when compared to the reference (top). The bottom trace displays the level of correlation between the sample and reference and mutations are indicated with high intensity peaks.
|
The HCV sample contains homozygous and heterozygous mutations, some altering the amino acid sequence.
|
Analysis Review Tool
Added to Mutation Surveyor version 3.30, March 2010, the review tool provides several methods to organize and review patient or other samples. The report is hyperlinked to the electropherogram window permitting rapid review and editing of found variant within the group.
Sequence Assembly
Download application note
For the genetic analysts working with sequences that do not have genomic references, Mutation Surveyor is a powerful tool capable of taking a group of sequences and assembling them into one long reference file. Just as the shotgun sequencing method, when used to sequence the human genome, required these short sequences to be assembled, many other projects require the same functionality. Mutation Surveyor is capable of assembling sequences of many formats including *ab1, *abi and *scf. The resulting text file containing a single nucleotide text string can then be utilized directly by the software as the reference file or annotated similarly to a GenBank file for identification of the coding sequence, variations and more.
In addition to its short sequence assembly tool, Mutation Surveyor is a powerful genetic analysis package capable of comparing samples to reference sequences and easily finding nucleotide and amino acid variations. All types of single nucleotide polymorphisms (SNPs) including homozygous substitutions, heterozygous substitutions with as little as 5-10% contribution from the minor allele and insertions and deletions (INDELs) can be detected with the click of only a couple of buttons. Mutation Surveyor provides an accuracy >99% in detecting homozygous and heterozygous SNPs when both forward and reverse traces are of Phred 20 quality. In addition, the complex heterozygous INDELs are deconvoluted from each other yielding two clean traces for easy identification of nucleotide variations.
 |
Mutation Surveyor will assemble overlapping sequences into one sequence text string. As shown in the figure the sample traces used to generate the assembled sequence are aligned properly to the assembled sequence.
Mutation Surveyor has tools to assist in improving the usefulness of the assembled sequence file including defining the coding sequence, setting primer sites, generating regions of interest, and selecting known variations. Assembled sequence files can be annotated to include the coding sequences, regions of interest surrounding each exon and known variations. The *seq file can now be saved as a *gbk file and opened in the GenBank Sequence File field when opening data to process. |
Samples traces overlap at ends and align. These samples were assembled to create a single reference *seq file shown in GenBank window. Within the Sample window, the traces are shown aligned to the reference.
Whole Gene Assembly
Assembly of a "whole gene" mutational analysis is easily accomplished with Mutation Surveyor. To assemble the mutational analysis of an entire gene, simply enter the GenBank data, pre-analyzed mutation projects, and the software will assemble and calculate an allele frequency, providing the locations of the found variants in bp order.
Reporting is extremely user-friendly with several customer customizable reporting formats, or customer can easily cut and paste areas of interest to any graphics capable program such as Microsoft Word. Reports can be easily exported in text, XML or HTML formats. Click here to see examples of the reporting feature.
Custom report builder
Mutation Surveyor v 3.2 and forward includes a custom report builder that provides users to “build” reports to meet their own specific needs. Customization features include methods to easily re-organize samples, remove mutation calls that are not of interest, while maintaining the data for future reference, change display information to meet specific requirements, color-code information for easy identification, quickly identify discrepancies within in sample groupings, organize output and mutation call by quality scoring add or create custom report header and importantly choose preferred nomenclature.
Data Organization
Mutation Surveyor provides point and click options to customize data presentation.
Analysis Presentation
1. Region of Interest
With a simple point and click users can “zero in” on regions of interest by hiding, not eliminating, mutation calls that fall out or the region of interest.
2. Color Coding
Color-Coding results allows users to highlight particular results for easy identification
3. Discrepancy Identification
Analysis anomalies are highlighted for quick identification and review user can easily switch between reports and mutation analysis for quick review and confirmation.
4. Normal Allele Scoring
|
|
|
|
Phred Score: 50
NM_Score: 20.5
High Score
Normal Allele
|
Phred Score: 20
NM_Score: 1.4
Moderate Score
Heterozygous
Substitution
|
Phred Score: 51
NM_Score: 0.0
Low Score
Homozygous
Substitution
|
Phred Score: 11
NM_Score: 5.2
High Score
False Positive
|
Mutation Surveyor provides a confidence check on the absence of mutations at each nucleotide greatly assisting in validating analysis results.
Download Genetic Diversity Application Note |
