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Using gap4 to check the trace_diff results

Trace_diff writes MUTN tags into the the readings' experiment file for each base it identifies as a possible mutation. Once a batch of suspected mutant readings have been searched by trace_diff and then assembled into a gap4 database the contig editor can be used to inspect the trace_diff results. The contig editor can produce displays like that shown below in which two traces are subtracted from one another and their differences plotted below.

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First the contig editor should be selected and configured for this type of operation. From the contig editor "Settings" menu (see section The Settings Menu) drag down to the "Trace display" line to bring up a cascading menu. First set the "Auto display traces" mode so that when searching for mutation tags (see below) the relevent traces will automatically be displayed. Next, if using a specific reading and trace that have been assembled into the database as a reference, then drag down to the "Diff against specific trace..." line to bring up a dialogue into which you should type the name of the reading. If using a consensus trace generated "on the fly" by the program drag down to, and set "Diff against consensus trace". For this mode you can also select "Only matching reads" to force the consensus trace to only use, at each point, only the traces from readings which agree with the consensus sequence. In addition, by selecting "Ignore selected read" you can specify that the reading being compared to the consensus trace is not included in the consensus trace calculation. Finally, from within the same menu you can set how the trace differences are displayed. If set, "Show positive differences" plots all differences upwards, but if not set, differences will be shown above and below the base line, as we have displayed them here. If "Y scale differences" is set the reference and mutation traces will be scaled in the vertical direction before subtraction so that the total signal at each point is the same. Usually we would switch off "Show positive differences" and "Y scale differences".

Once these settings are performed you can use the tag search function to search specifically for mutation tags (MUTN) which will step from tag to tag showing the relevent traces. This is set up from the "Search" item in the "Commands" menu, and once set you simply click on "Next search" to move to the next mutation tag.

There are several other 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. As mentioned above the editor search command (Commands menu) has a `tag' search method can be used to look through all tags of type MUTN. 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 Searching by discrepancies. Note that at present this search will only find at most one difference per column.

Once a mutation has been verified by trace checking, 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.


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This page is maintained by James Bonfield. Last generated on 2 Febuary 1999.
URL: http://www.mrc-lmb.cam.ac.uk/pubseq/manual/mutations_5.html