Several of the
Gap4 features can be used when looking for mutations within a
set of sequences.
For an overview of our mutation detection methods seesection Search for Mutations..
Before assembling into
trace_diff program should be used
to search for mutations and to tag the experiment files. This can be done
automatically by pregap4
(see section Pregap4 introduction).
Once the tagged mutations have been assembled into
Gap4 there are
several contig editor features available for checking the mutations. If
several sequencing runs from the same template insert are performed, the
`Group Readings by Templates' option (Settings menu) will reorder the
sequences so that readings from the same template are listed adjacent to
one another, regardless of their start positions within the contig. The
Settings menu also contains `Highlight Disagreements'
(see section The Settings Menu). This will
easily point out the bases which differ from the consensus at the base call
level. This will include both real mutations and those caused by base
calling errors. The editor search command (Commands menu) has a `tag'
search method can be used to look through all tags of type
This allows rapid checking of the candidate mutations found by
trace_diff. Further editor searches are planned to allow stepping
through tagged mutations in the order of best first. Currently if you wish
to check every difference (whether tagged or otherwise) the closest thing
available is the `discrepancies' search type, which can be used for
detecting any column where two or more bases above a specific quality value
(zero by default, implying all differences) are in disagreement.
See section Search by Discrepancies Note that
at present this search will only find at most one difference per column.
To verify the validity of a sequence difference the editor can show the traces
for the sequence, the wild type (if it's been assembled) and the differences
between the two. Alternatively the differences between the mutant trace and
the consensus trace can also be shown. The control of this behaviour and
the algorithms used for calculating the trace differences are adjustable in
the Trace submenu of the editor Settings menu.
See section Trace Display
The algorithm currently used by the
trace_diff program corresponds to
turning off both the `Trace difference algorithm' checkboxes.
Once verified, we recommend that you change the tag type (or create a tag if applicable) at that base position to facilitate easier searching in the future and to keep a record of which mutations have been manually checked.
Finally, note that for the purposes of printouts, the editor `Dump Contig to File' command saves sequences in the same style as currently displayed (except for tags). Hence using `Highlight Disagreements' will produce easily readable output.