Primer/Probe Interface

The Primer/Probe Interface provides information on individual reagents used for the typing of MHC or MHC-related loci. Users can access this information through the multiple functional frames within the interface. The words “primers” and “probes” represent SSOs, SSPs, SSP mixes, SSO mixes, and their combinations, which include nested PCR or SSO hybridization on group-specific amplifications. Nested PCR and group-specific amplification are both based on a primary amplification with an SSP mix.

Selection of Primers/Probes

The process of selecting a primer/probe begins by entering information in the upper frame of the Primer/Probe Interface page. Users enter search options for primers, probes, and mixes, which are grouped by type (SSO, SSP, SSO mix, and SSP mix), locus, and source (submitting institution). Primers/probes can also be selected by entering either the global or local name into the search field. Once this information is entered, the lower frame fills in with the Primer/Probe Listing. Primers/probes that are then selected by the user within the Primer/Probe Listing will be displayed in the large scroll box (in the upper frame) and can be downloaded in XML format.

Primer/Probe Listing

This function is found in the lower frame of the Primer/Probe Interface page. Results of a Primer/Probe selection are displayed here and will include a unique global ID, local name, source, locus, and type for each primer/probe result. The Global Name (global ID) of each result provides a link to the Primer/Probe View or Mix View functions, and the check box associated with each primer/probe/mix can be used to generate a list of items in the Primer/Probe Interface for subsequent download in XML format.

Box 2 contains information on global IDs.

Box 2

Global IDs.

Primer/Probe View

The Primer/Probe View is located in the upper frame of the Primer/Probe Interface and appears as a result of selecting a global ID. This view will display the entire set of data of the SSP and/or SSO primers and/or probes that the user had selected in the Primer/Probe Interface. The data displayed include:

• Local name
• Locus as specified by the submitter
• Global ID
• Date of last change (“last modified”)
• Probe orientation as specified by the submitter
• Corresponding allele sequence (Allele Seq:)
• Reagent type
• Optional filter, i.e., pre-amplification
• Version number
• Probe sequence (Probe Seq:)
• The annealing position of the 3' end of the probe as specified by the submitter
• Probe stringency

Primer/Probe View offers links that will enable a user to edit primer/probe information, change primer/probe stringency, and allow users to list alleles detected by a probe with a sequence alignment. If a list of alleles detected by a primer/probe without sequence alignment is wanted, Primer/Probe View can do this as well. Select Listed, and the results will be displayed in the Primer/Probe Allele Reactivities Listing.

Primer/Probe Edit

The primer/probe edit function allows users to enter new primers/probes into dbMHC or allows users to edit existing primer/probe data. All but the following three fields can be edited (these fields are set by dbMHC):

• Global ID
• Version number
• Date of last change

Within the primer/probe edit frame, there is an alternative input field for primer/probe sequence called Allele Sequence. If primer or probe orientation is set to “reverse”, the Allele Sequence field will reverse complement the allele sequence as displayed in the alignment to generate the appropriate primer/probe sequence.

Before submitting primer/probe data, a user should define the matching stringency of the primer or probe in the Annealing Stringency field. Once the user has selected the matching stringency, the probe can be submitted. Submission of a primer/probe triggers dbMHC to begin an allele reactivity calculation that is based on the primer/probe sequence and the selected matching stringency. The result of this calculation is a list of alleles that might be detected by the submitted primer/probe. The user will find this detectable allele list in the Reactive Allele Listing (lower frame).

Alignment, on the Primer/Probe View, opens a sequence alignment in the lower frame, which is the reactive allele alignment.

Reactive Allele Listing

The Reactive Allele Listing is located in the lower frame of the Primer/Probe Interface. It lists the alleles that might be detected by a selected primer/probe or mix (Boxes 3 and 4).

Box 3

Primer/probe reactivity.

Box 4

Submitter's reactivity scores vs. dbMHC reactivity scores.

Reactive Allele Alignment

Selecting Aligned from the Primer/Probe View results in a display of the reactive allele alignment in the lower frame of the Primer/Probe Interface. It displays the alleles that might be detected by a certain primer/probe or mix in alignment with that primer/probe sequence. By default, the first 20 alleles detected by the primer/probe or mix will be displayed. The frame for the reactive allele alignment also allows a submitter to set or edit the Allele Reactivity Score of a primer/probe in this frame. If a submitter chooses to not set a reactivity score, then dbMHC sets the submitter reactivity score to “not edited”. dbMHC will also calculate a system reactivity score for the primer/probe based on the primer/probe sequence and matching stringency. Colored option boxes indicate dbMHC's reactivity score for the primer/probe, whereas dots within the option boxes indicate the submitter's reactivity score. If the submitter's score (see Box 5) is set and differs from dbMHC's score, the dbMHC score box changes to a warning color.

Box 5

Setting allele reactivity scores.

The alignment position of the 3' end of the probe is recorded for each allele. Both the sense and the reverse alignment positions are displayed if the alignment represents an SSP mix.

Only users with permission from an account administrator will be able to edit or add additional alleles to a Reactive Allele Listing for a particular primer/probe or mix. To edit the allele reactivity list, mark the “edit reactivity list” check box in the reactive allele alignment frame.

Mix View

The Mix View function is located in the upper frame of the Primer/Probe Interface and can be accessed via the link on the global ID of the Primer/Probe Listing page.

It displays an entire set of mix data for selected SSO and SSP mixes:

• Local name
• Mix type
• Locus as specified by the submitter
• Optional filter, i.e., pre-amplification
• Global ID
• Version number
• Date of last change
• List of probes as mix elements
• Mix stringency

The Mix View function contains links to the Mix Element function, where users can change or add elements of a mix. Users will also find links to the Reactive Allele Listing, where they may list alleles detected by a selected mix or selected individual elements of a mix that do not have a sequence alignment. Finally, users will find links to the reactive allele alignment function, where they can view alleles with sequence alignments that are detected by a selected mix or selected individual elements of a mix.

Mix Edit

The Mix Edit function is located in the upper frame of the Primer/Probe Interface and allows users to enter new mixes or to edit existing mix data. Users can edit all but the following three fields (these fields are set by dbMHC):

• Global ID
• Version number
• Date of last change

Annealing stringency defines the cumulative matching stringency of the elements of the mix. For SSP mixes, both the sense and the reverse primer must react with the allele with at least the defined stringency.

Mix Element

The Mix Element function is located in the lower frame of the Primer/Probe Interface and allows users to add elements to a mix or edit existing elements of a mix. To use the Mix Element function, the user must first specify the mix to be altered in the Mix Edit function and define a source for the primers/probes that the user wants listed. The mix element function displays only SSO probes for SSO mixes and displays SSP probes from SSP mixes in a sense column and a reverse orientation column. For mixes that contain probes from different sources, users must enter the mix elements separately.

Selection of the Typing Kit

Use the Typing Kit Interface to enter search parameters for typing kits. Typing kits are grouped by:

• Type: SSO, SSP
• Locus
• Source: the submitting institution

Typing kits can also be selected by typing either the global or local name into the search field.

Search results, based on user-selected parameters, will be displayed in the Typing Kit Listing, which appears in the lower frame of the Typing Kit Interface. Typing kits selected from the Typing Kit Listing will be displayed in the large scroll box (of the upper frame). They either can be used for combined pattern interpretation of multiple kits or downloaded in XML format.

Typing Kit Listing

The Typing Kit Listing is displayed in the lower frame of the Typing Kit Interface. On the basis of the criteria selected, kits are listed with their unique global ID, local name, source, locus, and type. The global ID for each kit provides a link to the Typing Kit View (upper frame of the Typing Kit Interface). The check boxes associated with each typing kit are used to generate a list of items in the Kit Select view for subsequent interpretation of a pattern or for download in XML format.

Typing Kit View

The Typing Kit View (upper frame of the Typing Kit Interface) lists the entire set of primer/probe data for selected SSO and SSP kits. Typing kit probe data include:

• Local name
• Kit type
• Locus (with or without group-specific pre-amplifications)
• Global ID
• Version number
• Batch

If List Elements from the Typing Kit View is selected, the Typing Kit Elements list provides links to the kit components. Users will also find links to the Edit Kit Locus Groups and Elements page, where they can edit kit information, or they can use Save kit as... to access the New Typing Kit function, where they can create a new kit based on a currently displayed one (see Box 7).

Box 7

Creating a virtual kit.

Typing Kit Elements

Typing Kit Elements is displayed in the lower frame of the Typing Kit Interface and is used to display all of the elements (components) within a particular typing kit. This function will group the kit elements according to the kit locus groups and will display them as an ordered list. The global ID of each kit element serves as a link to either the Mix View or the Primer/Probe View of this element.

Edit Kit Locus: Groups and Elements

The Typing Kit Interface Locus: function (upper frame) can be considered a switchboard for assembling a typing kit for dbMHC, although kit name, batch, and type must be edited using the New Typing Kit function, accessed from New Kit on the Typing Kit Interface. The kit's global ID number, version number, and the date of last change are set by dbMHC. Users wanting to edit elements of a particular kit must select a particular locus first and then select Add locus group or Edit locus group, which will open the Edit Locus Group Elements page in the lower frame of the browser (see Box 8).

Box 8

Typing kit locus "groups".

Edit Locus Group Elements

The kit group function is located in the lower frame of the Typing Kit Interface and allows a user to add single elements to, or remove single elements from, a kit group. The kit group frame displays the elements for a particular locus selected by the user in the kit locus function. If the typing kit of interest is an SSO kit, only SSO or SSO mixes will be displayed. If the typing kit is an SSP kit, only SSP mixes will be displayed. Users can then add a primer/probe to the displayed group elements by clicking on a particular primer/probe in the left column and can remove a primer/probe from the group by selecting that element and clicking Remove.

New Typing Kit

Access to the kit edit function, New Kit, is found in the upper frame of the Typing Kit Interface and allows a user to enter or modify the name, batch, and type of typing kit. Users may change the kit type so long as the kit does not yet contain any element or group.

Typing Kit Interpretation

Access to the typing kit interpretation function, Interpret, is found on the Typing Kit Interface. It provides an online typing pattern interpretation tool. Typing kit reactivity patterns can be analyzed one at a time. Several typing kits that are used to type one sample can be analyzed in combination. An order number represents each element of a typing kit. This number provides a link to the probe reactivity alignment view of each element. The display also indicates whether, and for which elements, an allele-specific amplification has been used. Reactivity patterns can be entered either as a string or via the graphical display. Both the graphical display and the text field will update each other. The string entry accepts “1”, “+” for positive reactivity, “0”,“-” for negative reactivity, “n” for not tested, “w” for weak reactivity, and “?” for undetermined. The graphic entry symbols are green for positive reactivity, orange for weak reactivity, yellow for undetermined, white for negative reactivity, and gray for “not tested”. Users can preset the main reactivity by selecting one reactivity option and clicking on Set All. If the option cycle has been selected, repeated clicking on one reactivity field of a kit will cycle through all possible reactivities.

The reactivity string or pattern entered will be interpreted as a heterozygote allele combination. Multiple kits can be combined. Users can set the degree of tolerance, which limits the number of false-positive or false-negative reactivities per locus. Each locus is analyzed separately. Allele assignments for each locus are listed according to the number of false-positive/false-negative reactivities.

Typing Kit Pattern

Selecting Pattern from the Typing Kit Interface results in a display (in the lower frame) of a cross-tab view of allele reactivities of an individual typing kit. Alleles are listed in rows, and kit elements are listed in columns. Each element of a typing kit is represented by the respective order number. This number provides a link to the probe reactivity alignment view of each element. The display also indicates whether and for which elements an allele-specific amplification has been used.

A “+” with a green background signals a detection of an allele by a kit element, a “w” with an orange background signals a weak detection, a “?” with a yellow background signals a lack of information, and an “r” with a red background signals a rejected interaction, although originally suggested by the prediction algorithm. If a kit is designed to detect only a subset of alleles, the display will be limited to this subset (see Box 9).

Box 9

Some dbMHC/browser limitations.

Nucleotide Differences

dbMHC divides nucleotide interactions into five categories: perfect match, high match, medium match, poor match, and no match. dbMHC defines these categories by using the purine and pyrimidine interaction between the allele and the primer/probe, as well as the number of hydrogen bonds affected during the virtual pairing of the allele with the primer/probe. See Table 2.

Table 2

Penalties for nucleotide mismatches.

Nucleotide Position and Primer/Probe Type

dbMHC uses a penalty system based on nucleotide position in its calculation of SSO/SSP interactions with different alleles. The system dbMHC uses indicates the extent to which an individual nucleotide position will affect the interaction stringency grade in a worst-case scenario, where guanines are in opposing sequence positions or cytosines are in opposing sequence positions. The maximum penalty given in the system is in the middle of an SSO and at the 3' end of an SSP, as shown in Tables 3 and 4.

Table 3

Position penalties for SSP nucleotide mismatches.

Table 4

Position penalties for SSO nucleotide mismatches.

If the initial interaction stringency grade for a particular primer/probe–allele interaction is 100% and a mismatch occurs in a position that carries a penalty of 100%, then dbMHC will reduce the interaction stringency grade to 0%. If the mismatch occurs in a position that carries a penalty of 95%, dbMHC will reduce the interaction stringency grade to 5%. A more detailed example of how dbMHC computes primer/probe interaction stringency is available online.

dbMHC's reactive allele alignment algorithm searches for sequences with a match grade above the stringency level set by the submitter of each probe. Currently, the search algorithm searches within all loci that are part of dbMHC. Within each locus, the algorithm constructs compound sequences that represent the combined polymorphic positions of alleles of the same length. Alleles with insertions or deletions are handled separately. If a primer or probe matches with a certain position within the compound, all contributing alleles are checked for that position, whether or not they match.

The reactive allele alignment algorithm will check within a specified locus for sequences that have a match grade in accordance with primer/probe specifications at a user-indicated position. The algorithm then extends SSPs toward the 5' end of the probe, to a maximum of 10 nucleotides, and extends both sides of SSOs a maximum of 15 nucleotides, observing all polymorphic positions in matching alleles. The resulting probe extension is then used by the alignment algorithm to check for cross-reactivities within other loci.

If the submitted primer/probe sequence is shorter than 10 nucleotides and the user submits a score that accepts certain alleles and rejects others, the reactive allele alignment algorithm will use this information to refine the probe extension. If the probe submitter rejects all alleles containing a unique sequence motif in the vicinity of the probe sequence, the algorithm will generate the extended probe sequence such that it does not match the unique sequence motif.