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.
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.
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).
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".
(Click for full size image)
(Click for full size image)
Configuring The Gap4 Editor For Mutation Data
Using The Gap4 Editor With Mutation Data
Last generated on 25 November 2011.