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Mutation Detection Reference Data

The mutation detection methods require reference traces and optionally reference sequences. Reference traces are used for automatic mutation detection and for visual inspection of trace differences. Reference sequences are used in gap4 to provide a base numbering standard, and if required to provide feature table entries to control translation and mutation reporting.

Reference Sequences

Reference sequences are used in gap4 (see section Gap4 introduction). Here they can be used to define a numbering system independent of gaps introduced to produce alignments. The numbering can start at any point in the reference sequence. If the reference sequence is entered with a feature table the features are converted to tags and can be used to control translation of the sequence in the contig editor. For mutation detection work the reference sequence and feature table enable mutations to be reported using positions defined by the reference sequence, and also allows the effect of the mutations to be noted. Gap4 is able to store entries from the EMBL sequence library complete with their feature tables. These feature tables are converted to gap4 database annotations (tags), which means that they can be selectively displayed in the template display and editor, and used to translate only the exons (in the correct reading frame). Obviously it may be useful to augment the feature tables with the sites of known polymorphisms or deleterious mutations so that they can be displayed in gap4 as landmarks. When it comes to producing a report of the observed mutations the feature table is used to work out if a mutation is expressed and if so what the amino acid change is. Additional tags can be created to specify the positions of the primers or restriction sites used to obtain data covering segments of the sequence. For any project the reference sequence need only be set up once. Either project databases can be started with the reference sequence already configured or the reference can be assembled along with the reading data. The reference sequence can be designated (or reassigned) as follows. In pregap4 (see section Pregap4 introduction) it can be named in the module "Reference Traces". In the gap4 editor it can be set by right clicking on its name. Once set it should appear labelled "S" at the left edge of the editor.

Reference Traces

References traces are used by the automatic mutation detection program mutscan (see section Mutation Scanner, and by the trace difference display in the gap4 editor(see section Editing in gap4). Ideally forward and reverse reference traces should be available and should be obtained using the same primers and sequencing chemistry as the patient data. From the "settings" menu of the editor the trace display can be set to "Auto-Diff traces". Once this is activated, whenever the user double clicks on a base in the editor sequence display, not only is the reading's trace displayed, but also its designated reference trace plus the difference between them. If its complementary reading is available, its trace and reference trace and their differences are also displayed. These trace displays and the editing cursor scroll in synch.

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(Click for full size image)

Top and bottom strand differences for a heterozygous base.

The preferred way of assigning reference traces to readings is by use of "naming conventions"; that is to have a simple set of rules which control the names given to the trace files. It can be seen in the figures showing the editor that forward and reverse readings from the same patient have names with a common root but which end either F or R. This both ties the two together (so the software knows which is the corresponding complementary trace when the user double clicks on a reading) and also enables the association of readings and their reference traces. Once a convention has been adopted the rules can be defined for pregap4 by loading them via the "Load Naming Scheme" option in its File menu (see section Pregap4 Naming Schemes). For any batch of readings the reference traces are defined within pregap4's "Reference Traces" module. Note that this mode of operation, by allowing the specification of only one forward and one reverse trace, limits each batch of traces processed to those which correspond to a given pair of reference traces. The size of the batch is unlimited.

The alternative way of specifying the reference traces is to right click on their names in the editor. This also allows positive trace controls to be specified (which is not possible in pregap4).


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Last generated on 25 November 2011.