Overview Of Mutation Detection

Copyright (C) 1998-2003, Medical Research Council, Laboratory of Molecular Biology.

This document has been created to provide a brief overview to the current status of the mutation detection capabilities of the Staden Package.

We welcome comments.

Introduction to mutation detection

Our methods for detecting mutations are based on the alignment and comparison of the fluorescent traces produced by Sanger DNA sequencing. To use clinical terminology, samples from patients are compared to standard reference traces. Patient and reference traces should be produced using the same primers and sequencing chemistry, ideally from both strands of the DNA. The data shown in the examples below is from exon 11 of the BRCA1 gene.

New features in the 2003.0b1 release are described here.

The basic idea is illustrated in the following two figures which are screen dumps from our program gap4. The first shows a sample containing a point mutation and the second contains a heterozygous base position. The displays are bisected vertically: at the top left is the sample trace from one strand of the DNA, below that the reference trace for that strand, and underneath the difference between these traces which is obtained by subtracting one from the other. On the right is corresponding data from the other DNA strand.

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Figure 1. Top and bottom strand differences for a point mutation.

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Figure 2. Top and bottom strand differences for a heterozygous base.

As can be seen, although no vertical scaling is performed the difference trace is quite flat or is consistently either above or below the mid-line, except at the sites of mutations. Near these are strong peaks, but notice that only for the mutated base are there peaks both above and below the mid-line. The context effects caused by the mutation produce peaks only in one direction.

It is perhaps necessary to point out that analysis of the traces is essential because base callers make mistakes: they can assign the wrong base types and also assign single bases where the DNA is heterozygous. An example of the latter can be observed in Figure 2: on one strand the base caller has assigned a "-" symbol at position 251, at least indicating uncertainty, but on the other strand it has assigned "T". The DNA is clearly heterozygous at this position. This means that simply looking for differences between patient sequences and reference sequences will cause point mutations and heterozygous bases to be missed (of course base calling errors will also create false differences).

These trace displays alone are very useful for visual inspection of data and are all some users want. However we also have programs which automatically analyse the trace differences and tag the bases which have significant peaks as possible sites of mutation.

Trace viewing is initiated from within the gap4 editor. Each record in the editor shows an individual reading with its number and name at the left. Negative numbers denote readings which have been complemented. Several sequences have special status. At the top is a sequence labelled with a letter S at the left edge. This is the reference sequence, here the EMBL entry HSLBRCA1 which covers the entirety of the BRCA1 gene. The numbering at the top of the display corresponds to positions in this reference sequence. The program has also coloured (green) all exons on the reference sequence. The bottom DNA sequence in the editor is labelled "CONSENSUS". For mutation detection work this sequence is forced to be identical to the reference. Below the CONSENSUS sequence is the amino acid sequence for the reference. This is calculated on the fly using the feature table of the reference sequence and so translates only exons and in their correct reading frames. Two other sequences (near the top) are labelled R and F. These are the readings providing the reverse and forward reference traces for this segment of the data.

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Figure 3. A set of aligned sequence readings displayed in the gap4 editor.

At the very bottom of the editor is an information line which is used to display data about items touched by the mouse cursor. Here it is showing data about one of the positions tagged as possibly being heterozygous. It includes the observed base types (G and A) and the scores achieved by the automated analysis.

The editor can be set to show only differences between readings and the reference; all matching bases appear as dots. For example, Figure 4. shows the same data as Figure 3, but with the editor set to show differences, and the information line showing details about a possible mutation.

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Figure 4. An alternative view of aligned sequence readings in the gap4 editor.

One column contains several bases tagged in red, signifying possible heterozygotes, and some in orange denoting possible point mutations. During visual inspection the program can be made to move the cursor from one tag to the next and to automatically display the aligned traces as shown above in Figures 1 and 2.

It is also possible to have positive controls for displaying the trace differences; i.e. reference traces which contain the mutation. In this case the traces appear as shown in figure 5. Here the forward and reverse positive controls are shown to the right of the normal plots. In Figure 5 the positive control difference plots are quite flat hence, in this case, providing confirmation of the presence of the heterozygous base.

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Figure 5. Top and bottom strand differences and positive control for a heterozygous base.

As mentioned above the package contains programs which can automatically compare the traces and their reference sequences. The output from these programs are the tags shown in the editor. Users can check the traces at these positions using the displays shown in Figures 1, 2 and 5; if necessary removing or adding tags. Alternatively users can rely entirely on visual inspection and create all tags themselves.

Once all the mutations are correctly tagged the program can produce a report which includes the reading names, mutation positions relative to the reference sequence, the actual change, its effect, and the evidence. An example is shown below in Figure 6.

001321_11aF 33885T>Y (silent F) (strand - only)
001321_11aF 34407G>K (expressed E>[ED]) (strand - only)
001321_11cF 35512T>Y (silent L) (double stranded)
001321_11cF 35813C>Y (expressed P>[PL]) (double stranded)
001321_11dF 36314A>R (expressed E>[EG]) (double stranded)
001321_11eF 36749A>R (expressed K>[KR]) (double stranded)
001321_11eF 37313T>K (noncoding) (strand - only)
000256_11eF 36749A>G (expressed K>R) (double stranded)

Figure 6. How gap4 reports mutations.

Here the first record is for reading 001321_11aF, position 33885, T changed to T and C (i.e. is heterozygous) to produce no amino acid change, with evidence coming only from the complementary strand. The last record is for reading 000256_11eF, position 36749, A changed to G, producing an amino acid change K to R, with evidence from both strands of the sequence. The penultimate record denotes a heterozygote in a noncoding region.

Mutation Detection Methods

The software takes batches of trace data from sequencing instruments. It handles all processing except base calling (although it can employ third party programs such as phred for this step). This includes file conversions, quality clipping, scanning for mutations and heterzygotes, multiple sequence alignment, easy visual inspection of traces, production of reports, and the accumulation and storage of readings and traces. The software also handles the initialisation/configuration of standard reference files and databases for any project. The two main programs are pregap4 and gap4. Pregap4 prepares data for gap4 by automatically using a variety of smaller programs such as those used to search for mutations. Gap4 is used to store the aligned readings, to view the sequences and traces, and to produce a report listing the observed mutations.

Any number of sequences can be processed in a single run, and for each individual patient sample the operation is generally performed in two steps. First, via pregap4, the traces are aligned and compared to the reference traces and any possible mutations or heterozygous bases marked. Secondly, the data is transfered into a gap4 database from where users can visually check the differences between the reference and patient traces.

The description of the programs below is presented in reverse order of use i.e. gap4 then pregap4, but first we give further details about the use of reference data.

Mutation Detection Reference Data

The methods require reference traces and optionally reference sequences.

Mutation Detection Reference Sequences

In order to put readings and their mutations in context we use a reference sequence and feature table. This enables mutations to be reported using positions defined by the reference sequence, and also allows the effect of the mutations to be noted. To facilitate this 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 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.

Mutation Detection Reference Traces

References traces are used by the automatic mutation detection programs tracediff and hetscan, and by the trace difference display in the gap4 editor. 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. See Figures 1, 2 and 5. These trace displays and the editing cursor scroll in synch.

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. 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).

Automated Detetection Of Mutations and Heterozygous Bases

The package contains programs, tracediff and hetscan, which can automatically compare patient and reference traces to find point mutations and heterozygous bases. Comparison is performed by aligning the patient and reference traces and then analysing their differences. Users can set parameters which control the sensistivity of the algorithms (and hence which determine the ratio of false negative and positive results). Tracediff adds tags of type "mutation" to the patient files, and hetscan of type "heterozygous". The tags contain the numerical scores achieved at the site of the reported base changes, and they can be viewed via the gap4 editor. Tracediff and hetscan are normally run via pregap4.

Visual Inspection Of Mutation Data

The readings are stored aligned in a gap4 database. From here they can be viewed at three levels. The aligned sequences and traces can be displayed using the editor as shown in figures 1-5, and an overview obtained via the template display.

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.

Use Of The Gap4 Editor With Mutation Data

Although designed originally for handling shotgun sequence data the editor contains additional functionality for dealing with mutation data. Typical examples of the look of the editor are given above in Figures 3 and 4. From within the editor the trace displays can be requested and viewed.

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. 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. 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".

Processing Batches Of Mutation Data Trace Files

It is not clear which is the best way of organising the data for the simplest and most efficient processing using the current programs, but for now we make the following suggestions.

We assume that the region of the DNA being studied has a standard set of forward and reverse primer pairs covering all segments of interest and that a standard reference sequence in EMBL is available.

We recommend that batches of data from single primer pair combinations are processed separately, using separate temporary gap4 databases. For example, exon 11 of BRCA1 can be covered by five pairs of forward and reverse primers and we suggest that batches of traces obtained from each of these primer pairs should be processed using five gap4 databases.

Each processing run should create a new database and should enter, not only the new sets of patient data for that particular primer pair, but also the corresponding reference sequence and reference traces.

Obviously when several primer pairs are needed to cover a given region of the DNA (eg for BRCA1) the same reference sequence would be used for all the primer pairs.

An alternative to the above is to create a template database for each primer pair which contains the data for the corresponding forward and reverse reference traces plus the fully annotated reference sequence. These template databases are copied to create a temporary database for each new batch of data for the given primer pair.

Whichever of these two strategies is adopted each batch of new data is processed, analysed and assembled into these temporary databases, inspected visually, and a mutation report generated.

The use of separate temporary databases simplifies the assignment of reference traces and the use of the report generation function.

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Figure 9. An overview of a database containing data for only one primer pair of BRCA1

For long term storage and to facilitate larger studies, the content of each of these temporary databases is then transferred to archive databases, after which the temporary databases are no longer needed. The archive databases could be restricted to individual primer pairs or could accommodate data covering the whole of the reference sequence.

Processing Batches Of Mutation Data Trace Files Using Pregap4

All the data processing other than visual inspection of traces and report generation is handled by the program pregap4. Pregap4 achieves this by running a set of individual programs selected by the user.


Figure 10. The pregap4 Configure Modules window showing a typical list of mutation data option selections.

The "Configure Modules" window shown in Figure 10. is used to select which programs to apply to a batch of data, and to configure their usage. On the left is a list of programs and options, with "x" showing the ones that have been selected. If the user clicks on an option name its name is given a blue background and its configurable parameters are shown in the right hand panel to enable the user to alter them. Here "Reference Traces" has been selected which enables the user to set the reference traces and sequence.

The other selected options (marked with "x") are typical of the ones used for mutation detection studies. Below we describe the use of each plus a few alternatives. All of the options are descibed in more detail elsewhere in our documentation, our intention here is to give an overview of their use during mutation studies.

Note that the window labelled "Files to Process" is used to tell the program which files to process as a batch.

Configuration Of Pregap4 For Mutation Data

General Configuration
This option allows the user to select whether the trace names used for the samples should be the same as their file names or should be the names stored inside the files.
Phred is a base caller which also assigns confidence values to each base. Generally the data passed to pregap4 has already been base called. However not all base callers assign confidence values and so it can be useful to apply phred or ATQA (which does not base call but does assign confidence values). Alternatively "Estimate Base Accuracies" can be applied which is a simple program for providing numerical values which reflect the signal to noise ratio for each base, and which can be used instead of confidence values. (Note that if quality clipping is used, its score thresholds depend on whether confidence values of eba values are used).
Trace Format Conversion
This option can be used to convert bulky files such as those of ABI to a compact such as SCF or ZTR without loss of the data required for trace display.
Initialise Experiment Files
The input to gap4 and several of the other programs used here is a data known as Experiment file . This step, which has no configurable parameters is essential for mutation data processing.
Augment Experiment Files
The section on Reference Traces outlined the use of "Naming Schemes" for associating pairs of forward and reverse readings, and for assigning reference traces. The naming scheme must be loaded from pregap4's File menu. "Augment Experiment Files" must be activated in order for the naming scheme to be applied. No parameters need be set.
Quality Clip
The reliability of the base calls varies with position along the sequence. Near to both ends the data is less reliable. The "Quality Clip" option trims the ends of the sequences by analysing their confidence values or accuracy estimates (if present) or the density of unknown bases in the sequence. By observing these "clip points" other processing programs will work more reliably.
Reference Traces
As explained above it is necessary to specify a reference trace (preferably one for each strand of the data if processing data from both strands). The Reference sequence can also be set here. Note that even if our suggestion to preload the reference traces into the gap4 database is followed, it is still necessary to specify them here for use by the mutation detection modules.
Trace Difference
This is the program which compares the patient and reference traces to search for possible mutations. It adds data to the experiment files to mark each predicted mutation, and this data will appear as tags in the gap4 database. It can also create a new trace file containing the difference of the reference and the sample. The numerical parameters control the sensitivity of the algorithms, and hence the ratio between the numbers of false positive and negative resluts.
Heterozygote Scanner
This is the program which compares the patient and reference traces to search for possible heterzygous bases. It adds data to the experiment files to mark each predicted heterozygous base, and this data will appear as tags in the gap4 database. The numerical parameters control the sensitivity of the algorithms, and hence the ratio between the numbers of false positive and negative resluts.
Gap4 shotgun assembly
In order to be able report the positions of mutations relative to the reference sequence, and to be able to compare sets of samples from patients, it is necessary to perform multiple sequence alignment on the data. This is termed "assembly" and is usually performed by gap4, although other programs can be operated via pregap4. If following the suggestion to preload the reference sequence to a temporary database for each batch, supply the name of this database here. Otherwise a new database should be named and created from this option. (If this strategy is adopted make sure that the reference sequence and the references traces are assembled!) The parameters that control the assembly process and are described elsewhere.

Note that pregap4 has the facility to save its configuration and parameter settings. This means that the current configuration will be set automatically next time the program is used (and hence the steps just described only need to be performed once). In addition pregap4 can be run non-interactively by typing a single line on the command line. Taking thse two capabilities together, means that only one line need be typed in order to process all subsequent batches of data (assuming the file names are reused, which is easy to arrange.)

Discussion Of Mutation Data Processing Methods

The original version of these methods was described in James K Bonfield, Cristina Rada and Rodger Staden, "Automated detection of point mutations using fluorescent sequence trace subtraction", Nucleic Acids Res. 26, 3404-3409, 1998.. The more recent work has been done by Mark Jordan and James Bonfield.

At present pregap4 and gap4 clearly show their primary usage in the field of genome assembly, but versions tailored to mutation studies can be created once the requirements are agreed. Ideally all processing should be controlled by a single program which once configured for any project should require users to provide only the project name - all other file names and parameters could be preset, and all processing, including archiving and backup, performed automatically, leaving the data ready for visual inspection.

The automatic mutation and heterozygote detection programs work well on all the test data we have but now they require evaluation by external groups. Such analysis would enable us to improve the algorithms and to tune their parameters. At present we know that sometimes a base will be declared both as a mutation and as a heterozygous position when visual inspection shows that it is one or the other.

There is still much that can be done overall to improve the methods, but the text above summarises their status in July 2002. Although currently valuable for real scientific and clinical work they should perhaps be viewed as prototypes.

This page is maintained by staden-package. Last generated on 22 July 2002.
URL: http://www.mrc-lmb.cam.ac.uk/pubseq/manual/mutations_unix.html