CSCE-based Hetero-Duplex Analysis (HDA)

Hetero-Duplex Analysis (HDA) is an easy and cost-effective technique for rapid screening of mutations. DNA from target genes is amplified by PCR followed by a denaturation step. After renaturation, heterozygous mutant DNA or mixed wild-type and mutant DNA form heteroduplexes. This heteroduplex DNA has a different electrophoretic migration behavior compared to corresponding homoduplex DNA.

Known as Conformation-Sensitive Capillary Electrophoresis (CSCE), the hetero-duplex analysis principle has been refined to become a sensitive high-throughput mutation screening technique used on the automated capillary genetic analyzers from Applied Biosystems (Application Note 106IN02-01, May 2005). The degree of automation and the throughput capacity of these instruments as well as the accuracy of the data obtained allow large quantities of samples to be tested reliably for mutations, so that the sequencing workload can be reduced to the confirmation of potential mutants. CSCE has proven to be invaluable for the screening for hereditary diseases and cancers and offers great potential for many other applications in human genetics, plant breeding, bacteriology, etc.

In a typical CSCE experimental setup, a number of target genes are amplified to form one pool (panel) of fragments. In the simplest setup, the number of PCR products (targets) one can pool together is determined by the number of color dyes used. However, if PCR products with significantly different lengths can be designed, these can be pooled in one run using the same dye. The figure below shows a typical setup where 8 targets (PCR products) have been carefully chosen so that each time a short and a long fragment can be labelled with the same dye and mixed in the same pool.

Pooling strategy

HDA in BioNumerics

In collaboration with Applied Biosystems and three reference testing labs in the U.K. and the Netherlands, Applied Maths has built a highly specialized plugin for automated high-throughput CSCE based HDA in BioNumerics. The BioNumerics HDA plugin supports the use of combined PCR products with different length ranges (see example above) and has a built-in warning system for potential overlap between the ranges.

BioNumerics provides a fully automated workflow, from import of raw trace files to assignment of wild type or mutation type per amplicon. All results, problems or warnings during the workflow are reported in convenient overviews and can be viewed in full detail with a single mouse click.

Import of trace files

File name parsingNaming strategy is an important aspect in automation of the import process. BioNumerics can parse target and pool information from the trace file names when a consistent name formatting is used, but it can also read the information from the Sample and Comment fields provided in the trace files. Once the pooling and naming strategy is defined, batches of trace files can be imported automatically with just a few clicks. Although a batch will often consist of traces from the same run, the software offers the freedom to import and process traces from different runs as one batch.

Finding the right target peaks

Target PCRThe BioNumerics HDA software does not require internal size standards to be included in each trace, so that all available dyes can be used for sample data. Instead, target peaks are searched for within a defined area of the trace, using an expected position, a signal width and a left and right control range, all of which can be specified for each target individually. Even in setups where PCR products with different lengths are mixed using the same dye, correct target identification is always achieved once the parameters are set properly.

Automatic peak matching

Reliable peak matching is the most critical step in CSCE analysis. Applied Maths' expertise in pattern matching has resulted in a fast, sensitive, and reliable peak matching tool that can detect even the slightest deviations in peak shape. For each target PCR product, one or more target variants can be defined. For example, a wild type can be created, for which the user can define one or more representative peaks. In addition, polymorphic variants can be created, to which one or more representative peaks can be assigned as well.

The result of an automatic peak matching is displayed in an easy to interpret overview window. Reference peaks, matches, mismatches, failed peaks and potential problems are clearly indicated with text and intuitive colors.

HDA curves

HDA match resultA number of easy shortcut keystrokes and buttons or menu options exist to zoom in or out, generate detailed peak-to-peak comparisons, edit target information, arrange peaks according to similarity, etc.

Although the automatic matching algorithm can be tuned to be very sensitive and accurate using the right parameter settings, the matching results can easily be corrected for any individual peak. The user can change a matched peak into unmatched or an unmatched peak into a wild type or polymorphic variant. Peaks that were modified by the user are indicated in a different color. A comment field can be used to describe a particular peak or the changes made to it.

Since the assessment of the correct matching parameters is critical to the success of the automated pattern matching, the software provides special tools to automatically calculate optimal matching parameters based upon a set of known mutant and non-mutant samples. Calibration of the parameter set can thus be based on a batch of known samples entered into the software.

Databasing and reporting

The comprehensive and powerful databasing tools of the BioNumerics software allow CSCE samples to be described and documented with numerous information fields, attachments, pictures, HTML links, documents, etc. and to be used in a certified setting. On top of this, BioNumerics offers powerful querying and database viewing tools, so that even large databases containing many thousands of samples can be managed easily.

All samples from a batch, or any selection of samples from the database, can be brought together in a report window, displaying the matching results along with sample data of choice in a conveniently arranged overview. From this window too, peaks can be edited by the user and detailed peak-to-peak comparisons can be displayed with simple mouse-clicks or keystrokes.

HDA report

This plugin is license-based. Please contact us for licensing information.

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