The Biologist Friendly software

GeneMarker® is unique genotyping software with application oriented defaults that dramatically reduce analysis set-up time. Analysis is typically 3-clicks away from completion…PARAMETER SETTING…DATA SIZING…ANALYSIS REVIEW. The software’s robust sizing and pattern recognition technology automatically remove chemistry and separation artifacts.

GeneMarker has been designed to provide genetic researchers with a biologist friendly genotyping tool. We incorporated the suggestions and requirements of several research groups into the software. Their main requirements were ease-of-use, high accuracy, flexibility and low acquisition cost.

GeneMarker can perform analysis on up to 1,000 lanes of four or five color data sets generated by either slab gel or capillary electrophoresis. It is a unique genotyping tool as it is compatible with files from all major capillary and slab gel electrophoresis systems including ABI files (*.FSA, .*AB1, *.ABI), SCF files, MegaBACE™ files (*.RSD, *.ESD), SpectruMedix files (*.SMD, *.SMR), Beckman files, and slab gel image files (TIFF,BIP,JPEG and TXT) from such systems as the LI-COR DNA Analyzers and Kodak Image Station when used in combination with JelMarker.

GeneMarker is a replacement for such software packages as SAGA from LI-COR, TrueAllele from Cybergenetics, GeneMapper, Genotyper, and GeneScan from Applied Biosystems, MegaBACE Genetic Profiler and Fragment Profiler software.

The basic operation of GeneMarker automatically corrects for most instrument and chemistry errors, such as saturated peaks, noisy data, wavelength bleed-through, instrument spikes, and stutter peaks. GeneMarker’s automated Run Wizard is designed to make repetitive analysis quick, easy, and accurate.

User Operation
Sizing Technologies
Panel Selection

NEW! Merge Project – provides complete genotype by combining results of two or more multiplexes

Automated Applications, although GeneMarker can be used for any genotyping application, we have incorporated preset analysis modules for many key genotyping applications:

MLPA® (Multiplex Ligation‐dependent Probe Amplification)
MS‐MLPA®
Trisomy Analysis
MSI (Microsatellite Instability) Analysis
Kinship Analysis of Wild Populations and data base
AFLP
t‐RFLP
Microsatellite (SSR, STR, VNTR)
SBE/ SnapShot®
Cluster Analysis
TILLING® & EcoTilling with slabgel or capillary data
SNPWave®/SNPlex®
Haplotype Analysis

User Operation

The basic operation of GeneMarker automatically corrects for most instrument and chemistry errors, such as saturated peaks, noisy data, wavelength bleed-through, instrument spikes, and stutter peaks. GeneMarker’s automated Run Wizard is designed to make repetitive analysis quick, easy, and accurate.

Nearly every function of GeneMarker has been automated so that once the template is selected from the menu, or created by the user, the software automatically performs the analysis, providing a myriad of display and reporting options. Once the analysis has been completed and confirmed the software saves all of the analysis parameters, raw data and results for easy archiving and call back at a future date.

GeneMarker's wizard simplifies parameter setting by "walking" the user through 3 simple steps. Templates can either be selected from several embedded templates or the user can create and save additional templates; detection settings are presented in one dialogue box that creates required flexibility in a simple to use format. Panel management and creation requires only a few mouse clicks! Once set the parameters can be saved and called back for future analysis.

Sizing Technologies

As Separation technology is now achieving fragments in excess of 1000 base pair we have incorporated a new sixing technology in addition to the traditional Local Southern, Cubic Spline and third order least squares for the middle fragments. These older sizing methods have significant difficulties obtaining accurate size calls for data in excess of 800 bp, due to the non linear migration of larger DNA fragments.

Our “Large Size” sizing technology provides accurate, linear sizing using a DNA derivative migration time correction on fragments range from 30 to 1400bp. achieving resolution of 1base pair.

The “Large Size” technology affords many time, cost reducing multiplexing opportunities, such as the addition of 4x probes to clinical assays such as MLPA,Allele Specific Amplification (ARMs™, ASA, OLA, etc.) forensic profiling, and ecology analysis including TILLING®, STR/microsatellite profiles, AFLP, T-RFLP, VNTR and BAC fingerprinting.

Figure illustrates the non-linear DNA migration of GeneScan®1200 fragments in a POP7™gel. The migration time is linear for 100-800 bp fragments. The larger and smaller fragments deviate from the linear function. GeneMarker’s new algorithm provides accurate, linear sizing of the data using a DNA derivative migration time correction to large DNA fragments. Current sizing separation technology has its optimum efficiency at 470 bp. GeneMarker’s Large Size algorithm enables accurate sizing from small to large (30-1400) base pair fragments. The peak area in base space accurately determines the copy numbers because the peak area normalization is much less variable than that of the height normalization. The peak width is less than 1 bps for all fragments less than 1k.

Local Southern Algorithm has a limited linear range	New GeneMarker Algorithm provides linear range from 20–1200 bps

Comparison of the linear range for DNA fragments before with Local Southern (80‐800 bps) and after with GeneMarker’s Large Size Technology. The migration times are almost a perfect linear function to the DNA fragment size after applying the Large Size call in GeneMarker.

Download application note

Preloaded and Custom Panels

Panel templates can be selected from several embedded templates, downloaded and imported
from http://www.softgenetics.com/downloads.html or the user can create and save custom
templates. Previously saved ABI panels can also be imported from GeneMapper. Custom panels
are easily created in the panel editor; Automatically: using signal information from data files or
Manually: by simply inserting the desired alleles using the mouse.

Custom Panels are easily created for applications such as AFLP or microsatellite analysis.

MLPA® Analysis

Multiplex Ligation-dependent Probe Amplification is a simple method for simultaneous quantification of up to 45 nucleic acid sequences in a single reaction. Amplification products are separated by sequence gel electrophoresis, and MLPA® probes are able to distinguish between sequences that differ in only one base pair. Although the technique is efficient, inexpensive, and simple, there is a lack of an integrated software analysis package that performs data collection, normalization and patient reporting.

GeneMarker’s MLPA® function integrates all the analysis steps into a single convenient package. New analysis and reporting functions have been added to GeneMarker to increase analysis speed and reporting capabilities. GeneMarker is compatible with electrophoresis systems worldwide, including ABI files, MegaBase files, and SpectruMedix files, as well as slab gel output. With GeneMarker’s new application, copy number changes and loss of heterozygosity, which are key factors in the study of human cancers, are readily identifiable.

The software features pre-made panels based on the MRC Holland probe sets. These pre-made panels can be imported into the panel editor and will need minor adjustments to fit the panel to your experimental data. To access these panels, go to your computer’s Program Files, select the folder “SoftGenetics”, select GeneMarker, and finally open the folder “MLPA® Panels.” You may select from the list of panels including P035-DMDn, P087-BRCA1, and P023 DiGeorge Syndrome panels among the many others. Updated and new panels are available on the download page of this website.

Data Normalization

Due to the variations of PCR efficiencies from small to large DNA fragments or from sample to sample, two selectable normalization methods are provided. The first normalization method is the traditional method based upon the control probes as described in reference 4. The second method, unique to GeneMarker, normalizes peak intensities based upon the statically most probable median Figure illustrates the non-linear DNA migration of GeneScan®1200 fragments in a POP7™gel. The migration time is linear for 100-800 bp fragments. The larger and smaller fragments deviate from the linear function. GeneMarker’s new algorithm provides accurate, linear sizing of the data using a DNA derivative migration time correction to large DNA fragments. Current sizing separation technology has its optimum efficiency at 470 bp. GeneMarker’s Large Size algorithm enables accurate sizing from small to large (30-1400) base pair fragments. The peak area in base space accurately determines the copy numbers because the peak area normalization is much intensities. In order to correct for the peak intensity variation over size, an exponential function a*e-bz is used to fit to the square root of peak intensities, where z is size, and a and b are fitting constants. The normalization using the control probes is shown in figure 2. This correction removes the trend of dropping intensities as the DNA fragment size increases, and sets the height ratios of control probes to approximately 1. However the trend of peak intensities vary greatly from one sample to another with the internal control probes. We have found that the use of fewer control probes often results in large errors in the intensity normalization.

Normalization Methods

Traditional Control Probe Normalization.	SoftGenetics’ Population Normalization.

SoftGenetics’ “Population Normalization” addresses the above problems. Median peak intensities are derived from the first five data points, then sliding to data points 2-6, 3-7, etc. to ascertain the local median intensities. Outliers are rejected after applying a median filter. All of the probes (control and test) with the median intensities are then used to fit the exponential function. This methodology creates higher accuracy and lower false positive rates.

Customizable Patient Report available as a .PDF or printed file.

The patient report includes sample ID, analysis parameters, report, graph and electropherogram by sample.

Download application note (PDF)

MS-MLPA®

GeneMarker DNA analysis software has been successfully paired with the Multiplex Ligation‐dependent Probe Amplification (MLPA®) technique for detecting genetic deletions and duplications in various diseases including cancer. Recently, the technique has been improved to detect methylation sites within promoter regions and for genomic imprinting applications (5).Promoter Methylation kits from MRC‐Holland include ME001B Tumor Suppressor, ME002 Tumor Suppressor, and ME011 forMismatch Repair genes. Genomic Imprinting kits from MRC‐Holland include ME028 PWS‐AS and ME030 BWS‐RSS. GeneMarker’s new Methylation Specific – MLPA® module quickly and accurately detects methylation sites for researchers studying promoter methylation and genomic imprinting diseases. GeneMarker’s ease of use and professional reporting options are an excellent choice for MS‐MLPA® applications.

Methylation of the promoter region of tumor suppressor gene ESR1 is indicated by the red plot point that appears above the methylation threshold line.

The image on the left is a normal patient sample. Five genes of interest (methylation specific) from the PWS/AS region of chromosome 15 appear in the “Normal” region between the methylation detection threshold lines. The image on the right is a sample from a PWS patient. The five genes of interest are in a 1:1 ratio with the reference trace indicating methylation at these five sites (one copy of 15q11.2 and one copy of 15q12 have been deleted).

Download Application Note (PDF)

MLPA is a registered trademark of MRC-Holland B.V.

Trisomy Analysis

Full trisomy of an individual occurs due to non‐disjunction during meiosis I or meiosis II of gametogenesis resulting in 24 vice 23 chromosomes in a reproductive cell (sperm or egg) (1). Thus, after fertilization, the resulting fetus has 47 chromosomes versus the typical 46. The most common forms of autosomal trisomy are trisomy of chromosome 21 which results in Down Syndrome and trisomy of chromosome 18 which results in Edwards Syndrome. In rare cases, a fetus with trisomy of chromosome 13 can survive. Trisomy 13 is called Patau Syndrome. Autosomal trisomy is frequently associated with severe congenital abnormalities, mental retardation and shortened life expectancy. Aneuploidy of sex chromosomes can also occur: The presence of extra X chromosome(s) causes Klinefelter syndrome in men and Triple X syndrome in women, while monosomy X (45, X) gives rise to women with Turner syndrome.

Reporting options conform to Best Practice Guidelines (Dec. 2007). Download Aneuploidy Analysis Application Note

Reporting options include:

1. Use of peak height or peak area ratios
2. Identification of peak ratios that are consistent with designated triallelic (trisomy) range
3. Identification of peak ratios that are consistent with intermediate (inconclusive) ranges
4. Linear correction of data to resolve PCR bias
5. Display of ratio plots for population or individual sample<
6. Conclusion/Authorization table
7. Header automatically populated with info on sample ID, analysis parameters and analyst name/affiliation

GeneMarker facilitates accurate, rapid analysis of data generated by major QF-PCR kits such as: Aneufast™, Devyser™ and Elucigene™ and is compatible with genetic analyzers: ABI (Life Technologies), MegaBACE® (GE Healthcare), and CEQ (Beckman-Coulter).

Patient Report: Highlighted samples are consistent with user specified triallelic range and samples marked with a ‘?’ are consistent with user specified intermediate (inconclusive) ranges

GeneMarker has been designed to accurately detect trisomies using short tandem repeat markers derived from PCR DNA fragments.Trisomy individuals will either show three fragments of equal intensity or two fragments at a 2:1 or 1:2 ratio.

Ratio 1:2	Ratio 2:1	Ratio 1:1:1

The GeneMarker Trisomy tool offers two answers to the 2:1 trisomy detection question.

First, in the Ratio Plot in the bottom left corner of the analysis window, the peak intensity ratio of all markers are plotted. A linear regression line is run through the center of the data points and is used to correct for intensity drop due to fragment size increase. The Ratio Plot can be viewed as a linear regression plot or corrected for slope. This method of data correction aids in the detection of 2:1 ratio trisomies.

Report option provides display of ratio plots corrected for slope

The second aid in trisomy determination is the trisomy score. First a t‐value is determined and defined as the difference between the sample and the expected value divided by the standard deviation. There are two possible t‐values for every marker, one is the tvalue for heterozygote and the second is for a trisomy. T‐Score is the ratio of the heterozygote t‐value divided by the trisomy t‐value. Therefore, as the T‐Score increases, the confidence of the trisomy call also increases. A T‐Score greater than 5.0 is a confident trisomy call. A T‐Score less than 0.3 indicates a confident heterozygous call. Click here to Download Trisomy Application Note discussing linear regression and t-Score calculation/interpretation

Download Trisomy Application Note (PDF)

Download Aneuploidy Analysis Application Note (PDF)

Haplotype Analysis

Familial DNA fragment data is used for haplotype analysis in areas such as genetic disorder research and preimplantation studies. In GeneMarker, the Haplotype Analysis application combines allele call information from
multiple kits to obtain a complete genetic profile for each individual. The ‘Family Group Tool’ assists researchers
by automating the process of drawing pedigrees. The software uses the allele calls of children and parents to assign a first-order-approximation phase of the alleles from the familial data. Whenever the alleles are informative for phase assignment a pattern/color bar is assigned to indicate most probable phase.

Results (figure 1) X-Linked and Autosomal Linkage Examples

X-linked Example: Personal information Phase assignment Carrier status Standard symbols for male, female, Miscarriage (SAB)

Autosomal linkage Example: Phase assignment Cross-over in child 1 Standard symbols for male, female, deceased, pregnancy

Features of GeneMarker’s Haplotype Analysis Application
1. Follows Bennett et al. nomenclature for pedigrees
2. X-linked and autosomal pedigree formats based on parent/child(ren) allele calls
3. Edit family and individual information
4. Displays markers, allele calls, personal information in pedigree
5. Control ordering of markers in pedigree by customizing panels
6. Automatically makes first order phase assignment
7. Edit capability for reassigning phase or edit crossover
8. Save pedigree and re-open for adding data and editing
9. Allele conflicts flagged; detects potential uniparental disomy

Download Haplotype Analysis Application Note (PDF)

Microsatellite Instability (MSI)

Microsatellites are stretches of DNA where a 1‐5 base pair sequence is repeated several times. The most common microsatellite in the humans is a dinucleotide repeat of CA which occurs tens of thousands of times across the genome. Microsatellite instability (MSI) is a condition where repeat units are gained or lost within a locus resulting in length polymorphism. Certain repeat regions are known to be highly polymorphic and hereditable. In the forensics community, these sites are useful when identifying markers for DNA fingerprinting. Conversely, microsatellite instability within and around certain genes can have devastating effects due to the possibility of frameshift mutations.

In GeneMarker’s MSI Analysis module, tumor samples are compared to normal samples based on peak‐to‐peak comparison. Differences between the two traces are displayed in a Trace Comparison Histogram below each electropherogram. Within the electropherogram, the tumor sample trace is overlain on a light red reference trace. In this way, the clinician can easily visualize where the areas of instability exist. There are a few options for MSI display which will vary by personal preference. In the MSI Analysis Settings box there are several options for filtering the allele call. The peak detection thresholds, Intensity and Local Region are designed to filter out noise near the baseline. The Stutter Filter right and left thresholds are applied after the peak to‐peak comparison with the reference trace and are designed so that the user can minimize the number of bars in the Peak Comparison Histogram.

GeneMarker’s MSI Analysis Report

Download MSI Application Note (PDF)

Kinship Analysis and Data Base

GeneMarker now includes a first of its kind Kinship Analysis and data base module which enables ecologists, and other wildlife researchers to easily identify kinships in natural populations, livestock breeding programs and aquaculture.

The "Kinship Analysis" tool provides a report table with probabilities and likelihood ratios across three generations for sample pairs. The rigorous statistical analysis to determine levels of kinship uses identity by descent (IBD), follows the methods of Brenner and uses stochastic matrices of Li and Sachs. GeneMarker database search tool identifies samples with the same STR profile and calculates the random match probability (the probability that a randomly selected individual from a population will have an identical STR profile at the DNA markers tested). The "Find Family" tool searches the database and identifies files with the highest likelihood ratio for each relationship level to the experimental sample. Genetic Analysis Parameters allow setting tolerances for mistyping or mutation. The save to database function in GeneMarker can accept allele frequency tables for species specific markers and can accept previously archived genotype .txt or .cmf files, providing easy database updates.

GeneMarker’s Database Search Report: Report Settings to Customize Search for Animal or Plant Samples

Kinship Analysis Settings Provide Customization of Report Format

Download Application Note Kinship and Database Search: Animals

Download Application Note Kinship and Database Search: Monoecious Plants and Invertebrates

Download Plant & Animal Genome Kinship Poster

AFLP Analysis

Amplified Fragment Length Polymorphism (AFLP®) is a polymerase chain reaction (PCR) based genetic fingerprinting technique developed in the early 1990’s by Keygene. AFLP uses restriction enzymes to cut genomic DNA, followed by ligation of complimentary double stranded adaptors to the ends of the restriction fragments. A subset of the restriction fragments are then amplified using 2 primers complimentary to the adaptor and restriction site fragments. The fragments are visualized on denaturing polyacrylamide gels through either autoradiographic or fluorescence methodologies.

The AFLP technology has the capability to detect various polymorphisms in different genomic regions simultaneously. It is also highly sensitive and reproducible. As a result, AFLP has become widely used for the identification of genetic variation in strains or closely related species of plants, fungi, animals, and bacteria. The AFLP technology has been used in criminal and paternity tests, in population genetics to determine slight differences within populations, and in linkage studies to generate maps for QTL analysis.

GeneMarker ® is an efficient, user-friendly software tool designed for the analysis of data generated by AFLP technology. The software is compatible with electrophoresis systems worldwide, including ABI (AppliedBioSystems) files, MegaBase files, andSpectruMedix files, as well as slab gel output. The software features high efficiency allele calling, adjustable parameters and various reporting options including a trace comparison report. GeneMarker’s unique sizing and pattern recognition technologies significantly impove analysis accuracy while providing greater analysis speed and less user intervention.

Comparison courtesy of Meghan Avolio,Yale University, Department of Ecology and Evolutionary Biology, New Haven CT 06520

GeneMarker’s New Large Fragment Sizing Technology expands and reduces the cost of the AFLP technique by affording never before multiplexing opportunities.

Report displaying presence and absence of alleles	Allele Report displaying peak intensities

Download AFLP Application Note
Download AFLP Comparative Analysis of Andropogon gerardii with GeneMarker® & GeneMapper® Software Packages

T‐RFLP (Terminal‐Restriction Fragment Length Polymorphism) Analysis

GeneMarker can perform fragment analysis and genotyping on four or five color data sets from any slab gel or capillary electrophoresis system. The software automatically corrects for many common problems—instrument spike,color pull‐up, peak pullup, noisy data, saturated peaks and stutter peaks—saving significant analysis time and cost, efficiently analyzing raw fragment data within seconds. GeneMarker is robust software to analyze DNA fragment data labeled with MegaBACE™ dyes (Amersham), Big Dye® (AppliedBiosystems Inc.) or Beckman dyes from a variety of platforms: ABI DNA Analyzer or Genetic Analyzer, Amersham instruments or Beckman instruments.

GeneMarker’s New Large Fragment Sizing Technology expands and reduces the cost of the AFLP technique by affording never before multiplexing opportunities.

Peak table displayed below a sample electropherogram showing the option menu for editing items in the peak table (clicking the right mouse button with the cursor on a highlighted cell will activate the editing menu).	Print Report: Comparison of electropherograms from four samples (only one sample from a duplicate pair is shown).

Microsatellite Analysis with Linked Pedigree

GeneMarker decreases analysis set-up time through automated correction of common genotyping problems including saturated peaks, noisy data, wavelength bleeding, instrument spikes and stutter peaks. GeneMarker’s automated Run Wizard is designed to make analysis quick, easy, and accurate. The Data Analysis window features include:

1. Saturation Correction: Analysis of saturated data points by creating a synthetic peak based upon the peak
   shape before and after saturation.
   
2. Baseline Subtraction: The software removes the baseline so that the Y axis is above the noise level.
   
3. Pull-up Correction: This function removes peaks caused by wavelength bleeding.
   
4. Spike Correction: The software automatically removes peaks from voltage spikes caused by micro- air
   bubbles or debris in the laser path.
   
5. Stutter Peak Correction: The software automatically filters for stutter peaks caused by PCR slippage.

Linked Pedigree Tool

The user can either open an existing pedigree file of type ped or pre or create a new pedigree file in GeneMarker’s pedigree tool. The pedigree chart is designed to aid identification of inheritance patterns and abnormalities. All individuals in the pedigree with sample files are directly linked to the corresponding electropherograms with a mouse click, and individuals with illogical or abnormal allele calls are highlighted in red. The link between the pedigree and the electropherograms displaying allele calls for each marker make analysis makes analysis quick and efficient.

Pedigree chart showing inheritance conflicts in Family 5005

Download Microsatellite Analysis Application Note

Single Base Extension/SnapShot®

GeneMarker is an excellent tool to determine SNP genotypes is single base primer extension or SBE. An unlabeled primer with its 3’ end directly flanking the SNP is extended one nucleotide by Taq polymerase and fluorescently‐labeled ddNTPs complementary to the polymorphic base are added. The resulting fragment is one nucleotide longer, but the observed fragment size on a gel will be greater than expected due to the influence of the fluorescent dye on the electrophoretic mobility of these small fragments. SNPs can be identified by the one‐ or two‐color peaks associated with the incorporated labeled ddNTP and the length of the primer.

GeneMarker is user‐friendly software containing a robust size calling algorithm that resolves fragment lengths to less than one base pair with high efficiency allele calling, adjusts for mobility shift of small fragments and displays two‐color SNPs on one trace, essential for data generated by SNuPE and SNaPshot. The software is capable of analyzing data files from all major capillary electrophoresis systems including ABI, MegaBACE and Beckman. GeneMarker, in combination with our JelMarkerTM can also be used for analysis of slab gel output.

SNP Analysis for SnapShot® Report

SNPlex® & SNPWave®Analysis

One high‐throughput method to determine SNP genotypes is SNPWave (Keygene N.V.). SNPWave uses multiplex oligonucleotide ligation amplification of allele‐specific probes coupled with AFLP®‐primer selective amplification. SNPWave can detect up to 100 SNPs.

Circularizing padlock ligation probes are constructed that are specific to the SNP and flanking sequences. Locus‐specific probes will hybridize to complementary denatured genomic DNA. Allele‐specificity is determined by the SNP at the 5’ end of the padlock probe. Probes that contain a 5’ nucleotide complementary to the SNP will be ligated and amplified by PCR in subsequent reactions. Probes that do not contain a 5’ nucleotide complementary to the SNP will not be ligated and will not be amplified. The padlock probes contain stuffer regions and primer binding sites for AFLP‐selective amplification. The ligated padlock probe is amplified using fluorescently‐labeled +2‐selective and unlabeled non‐selective AFLP primers. The stuffer region provides length discrimination between alleles and among loci. SNPs are separated by 2 base pairs and loci are separated by 3 bp. The fragments are separated by size using capillary electrophoresis. Fragment dye color and length indicate SNP locus and allele.

An alternate methodology is the SNPlexTM Genotyping System (Applied Biosystems) which can interrogate 48 SNPs simultaneously and has been used to investigate SNPs in 92 cancer‐related genes in breast cancer (5) and to genotype plants (6). GeneMarker genotyping software is designed for fast, accurate and efficient analysis of SNPlex data.

GeneMarker is an excellent choice for the analysis of data from either of these techniques.

GeneMarker’s SNPlex Report	GeneMarker’s SNPWave Report

Download SNPlex Application Note

Download SNPWave Application Note

Phylogeny Clustering Analysis

Biological applications of data clustering calculations include phylogeny analysis and community comparisons in ecology, gene expression pattern, enzymatic pathway mapping, and functional gene family classification in the bioinformatics field. It has been successfully paired with the AFLP, microsatellite and RAPD analysis techniques for a variety of applications.

GeneMarker uses the Hierarchical Clustering method, treating each data point as a single cluster and successively merges clusters until all points have been merged into a single remaining cluster. Researcher may select from different linkage types – Single, Complete or the Average – for the clustering algorithm. Results are presented as a dendrogram and a table providing Euclidian distances between each point.

Dendrogram from Cluster Analysis of 30 files using allele calls from one multiplex (left) and dendrogram of the same files based on the results of 3 multiplexes (right). Allele calls were combined using the Merge Project tool in GeneMarker prior to Cluster Analysis.

Download Cluster Analysis Application Note (PDF)

Comprehensive Genotype using Merge Project Tool

Obtaining a complete genetic profile for wildlife and plant research or medical research is often complicated by overlapping marker ranges and/or incompatible chemistry; making it necessary to amplify the same samples multiple times using different sets of markers or locus specific primers. Traditionally, researchers export the genotyping results from several multiplexes into a spread sheet and manually combine allele calls for each individual.

The Merge Projects tool allows researchers to conveniently combine two or more GeneMarker projects (each project using a panel containing a unique set of markers or loci) into a single, comprehensive report. This report represents a single view of multiplexes (a super panel) of markers/loci from the individual projects, providing a complete genotype for each sample. This merged report may be saved in a spread sheet as a genome-wide genotype, or imported into other GeneMarker special applications, such as, Clustering Analysis or Relationship Testing and Kinship Analysis; improving the robustness of these analyses by including information from a greater number of markers.

Three different multiplexes were used to amplify 30 DNA samples. Each multiplex contained primers for 4 or 5
independently assorting loci. The merge project tool in GeneMarker provided a single genotype for each individual
with 14 markers. Allele drop out is indicated by **.

Report style options:

• Marker Table suitable for further analysis in Relationship Testing or Kinship Analysis
  
• Peak Table provides a spread sheet with marker and allele name, fragment size (MW), peak height, height
  ratio, peak area, area ratio
  
• Bin Table for further analysis using clustering algorithms for phylogeny.

Download Merge Project Application Note (PDF)

TILLING®& EcoTilling of Capillary and Slabgel data

With the 2007 Science breakthrough of the year going to Human Genetic Variation the importance of methods for studying variations are at an all time high. The techniques of Targeted Induced Local Lesions In Genomes, or TILLING, and EcoTILLING have been widely used since 2000 to detect Single Nucleotide Polymorphisms (SNPs) in organisms ranging from Arabidopsis (2) to zebrafish (3). The test samples may be experimentally mutagenized (ethylmethanesulfonate, radiation, etc) or from natural populations or derived from tumors or diseased tissues.

Briefly, the genes of interest are identified with gene‐specific primers and PCR amplified. The amplicon’s primers are labeled with two fluorescent dyes – the forward is often labeled with FAM‐blue and the reverse primer is often labeled with HEX‐green. The samples are mixed so heteroduplexes can be formed. The hybridized fragments are cleaved at the heteroduplex site by CEL I or Surveyor™ Nuclease, generating multiple pairs of fragments of complementary length and dye color (4). The denatured samples can be run through gel electrophoresis or mixed with an internal size standard and run through capillary electrophoresis. SNPs will yield two peaks of different color and the sum of the sizes will equal the amplicon length.

When using GeneMarker’s Tilling Application module, the size standard peaks are identified and all sample peaks are aligned. The peaks are smoothed, baseline is subtracted, and lane intensities are normalized. Low quality data is automatically rejected. A synthetic reference trace (Synthetic Control Sample) is constructed using median peak intensities from all of the high quality traces. This reference is subtracted from each sample trace generating a Mutation Chart that automatically identifies the sample’s variations as shown in figure with the amplicon length of 1049bps.

GeneMarker TILLING Analysis

The top panel shows the Synthetic Control Sample obtained from the median intensity after peak alignment. The middle panel shows sample 79. The bottom panel shows the Mutation Chart, generated by subtracting the reference from the sample, identifying individual variations. A blue peak at 205.2bps and a green peak at 848.8bps have been automatically identified. The original amplicon size is 1049bps.

Download TILLING Application Note

Application Notes

• Complete Genotype with Merge Project Tool (PDF)
• Automatic Phase Determination of Disease Haplotype from Family Genotyping Data (PDF)
• Kinship Analysis and Duplicate Sample Identification of Animal Microsatellite Markers with GeneMarker® (PDF)
• GeneMarker® Software for Multiplex Ligation-dependent Probe Amplification (MLPA™) (PDF)
• Methylation Specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA®) with GeneMarker® (PDF)
• GeneMarker® Software for Trisomy Analysis (PDF)
• Loss of Heterozygosity Detection with GeneMarker® (PDF)
• Software for Amplified Fragment Length Polymorphism (AFLP®) (PDF)
• GeneMarker® Software for Terminal-Restriction Fragment Length Polymorphism (T-RFLP) Data Analysis (PDF)
• Microsatellite Analysis with Linked Pedigree Tool (PDF)
• GeneMarker® software for SNPlex™analysis (PDF)
• GeneMarker® Software for SNPWave™Analysis (PDF)
• Clustering Algorithms for Genetic Analysis with GeneMarker® Note (PDF)
• Analysis of MegaBACE™ Data with GeneMarker® Software Application Note (PDF)
• Capillary Tilling Application Note (PDF)