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Using The Template Display With Mutation Data

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Figure 7. The template display showing the whole of the BRCA1 gene (exons in green).

The view obtained from the Template display and shown in Figure 7 is not of practical use but serves here to illustrate the overall arrangement of the data for our chosen example the BRCA1 gene. This figure shows the entirety of the EMBL entry HSLBRCA1 with its exons marked in green. Only exon 11 has patient trace data stacked above it.

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Figure 8. A zoomed-in version of the data shown in Figure 7.

Here we can see all the readings covering exon 11. Forward readings are light blue, reverse readings orange, primers are marked in yellow, mutations in red and orange. A common mutation appears in the leftmost set of readings and illustrates the value of using the template display for visualising the overall pattern of the tagged mutations.

Configuring The Gap4 Editor For Mutation Data

The current version of the gap4 editor contains very many options that are not needed for mutation data. Given sufficient demand a version tailored for mutation studies could be produced. For now it might make it easier to understand the program if its origin as a genome assembly program is borne in mind. Here we outline the options and settings relevant to mutation studies. The assignment of reference sequence and traces is described above. From the editor they can be set by right clicking on the reading names.

Gap4 enables segments of sequences to be annotated (or tagged). Each tag has a type (eg primer) and each type has an associated colour. Each instance of a tag can include editable text. This text can be viewed and edited by right clicking on the tag and selecting "Edit tag", after which a text box will appear. Gap4 can display annotations/tags as background colour and the user can specify which tag types are shown. For mutation studies the following tag types may usefully be activated, and all others turned off. Using the "Set Active Tags" option in the "Settings" menu first click on "Clear all". Then click on "primer". To add further types you must hold down the "Ctrl" key on the keyboard while clicking. Now scroll down and click on "Mutation", "Heterozygous" and "FEATURE CDS". Add any others required, then click "OK".

The following configurations are performed via the "Settings" menu.

Gap4 has three consensus generation algorithms. When using a reference sequence it is convenient if the consensus shown in the editor is forced to be the same as the reference. This will be the case if either the "Weighted base frequencies" or the "Confidence values" consensus algorithms are being used. This selection is made using the "Consensus algorithm" option.

Translations are shown in what gap4 refers to as the "Status" line. To enable automatic translation of the exons defined in the reference sequence, in the "Status Line" option set "Translate using feature tables".

To enable automatic display of trace diferences, in the "Trace Display" option set "Auto-Diff Traces".

To show only the base differences between the consensus/reference, set "Highlight Disagreements". These can be shown by dots or colour.

To show base confidence values set "Show reading quality" and also make sure that the value in the box labelled "Q" at the top left of the editor is set to 0 or greater.

To force forward and reverse reading pairs to be shown in adjacent records in the editor set "Group readings by templates" (NB this assumes that an appropriate naming scheme has been used).

If a reference sequence is assigned, the numbering at the top of the sequence will reflect the base positions in that sequence. Any pads in the reference sequence are ignored. If no reference sequence is assigned, the numbering will ignore pads if the "Show unpadded positions" option is activated.

At the bottom of the "Settings" menu is an option to "Save settings". Use of this will mean that the current configuration will be set automatically next time the editor is used (and hence the steps just described only need to be performed once).

Using The Gap4 Editor With Mutation Data

The current version of the editor has a fixed width and a maximum height. If too many sequences are present at any position a vertical scrollbar on the right edge can be used to move them up and down. The CONSENSUS line will always be visible, but at present, the reference sequence is scrolled along with all the other sequences and so may disappear. Horizontal scrolling is achieved in the usual ways, plus by use of the >, >> and <, << buttons. The reading names can be moved left and right using the scrollbar above them.

Configure the editor as described above.

The traces for readings (and their reverse) can be examined over their full length one at a time by simply double clicking on them then scrolling along. Any mutations observed can be labelled by right clicking on the base in the editor display and invoking the "Create tag" option. This brings up a dialogue box. At the top is a button marked "Type:comment"; clicking on this will bring up another dialogue with a list of all the tag types; choose the appropriate one ("Heterozygous" or "Mutation"). There are obviously many advantages to examining the traces like this using gap4. However, if the automated mutation detection methods are trusted, or used in way that makes them trustworthy for the type of study being undertaken, then there are quicker ways of examining the data.

The "Next Search" button at the top of the editor gives access to many types of search, one of which is "tag type". If this is selected a button appears labelled "Tag type COMM(Comment)". Clicking on this will bring up a dialogue showing all the available tag types. If the user selects, say "Mutation", each time the "Next Search" button is used the program will position the editing cursor on the next mutation tag. Double clicking will automatically bring up the appropriate traces as shown in figures 1, 2 and 5 (see section Introduction to mutation detection). The user can view the traces and if necessary alter the tag (eg delete it if it is a false positive).

Once all the data has been checked and all mutations and heterozygous bases have been tagged a report can be generated using the "Report Mutations" option in the editor "Commands" menu. Note that it is also possible to simply report all differences between base calls and the reference, but the usual procedure is for the program to report all bases tagged as "Mutation" or "Heterozygous". Example output is shown above in Figure 6 (see section Introduction to mutation detection). The report appears in the gap4 "Output window" which can be saved to disk by right clicking on the text and selecting "Output to disk".


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Last generated on 25 November 2011.