Created by Damian Goodridge Page 1 of 38 Created on 12/10/2004 2:08 PM. User Guide. Assign-SBT TM 3.2.7

Transcription

1 Created by Damian Goodridge Page 1 of 38 User Guide Assign-SBT TM 3.2.7

2 Created by Damian Goodridge Page 2 of 38 1 Introduction Overview Unique Features Summary of Functions Getting Started Installation Operator Login Changing Your Password or Default Settings Adding New Users Encoding The Analysis Window Setting the parameters for sequence analysis Settings Dialogue Box - Summary of Functions Sample Delimiters and Sample Naming Conventions Creating Locus Codes The HLA Locus Prompt Primer Site Trimming Matching Mode Library Mode Heterozygous Ambiguity Resolution Primers (HARP) Display Automated Editing Updating the Settings Sequence Editing and Allele Assignment Importing sequences for analysis The Analysis Window The Test Sample Pane The Sample Menu The Sequence Pane The Reference Consensus Mismatch Positions Confirmed Positions Edited Positions The Sample Consensus Electropherograms (EPG s) The Editor Bar The Assignment Pane Sequence Editing The Navigator Current Position Information Search Filters Position Selection Sample Selection Base Selection Layer Selection Resolution of Heterozygous Ambiguities by DNA sequencing...28

3 Created by Damian Goodridge Page 3 of 38 6 Reports The Report Generator Report Type Output Selection Sort Order Output Filters BCS Analysis Selection BCS Report FASTA file Output Format Database submission Generate Report Saving / Closing / Opening Appendix Menu Functions Sequence Libraries and Assign SBT TM 3 Applications Consensus sequence symbol definitions How Assign SBT TM 3 Handles Insertions and Deletions Contact Us:... 38

4 Created by Damian Goodridge Page 4 of 38 Compatibility Computer Operator Systems Assign SBT TM 3 is designed for use with Windows NT, Windows 2000 and Windows XP. Microsoft Excel 97 or above is required for the creation of enhanced reports. DNA Sequencing Chemistry and DNA Sequencers Sequences from Amersham DYEnamic ET Terminators, Applied Biosystems Big Dye Terminators have been analysed with Assign SBT TM 3 Assign-SBT TM 3 is designed for use with.ab1 or.abd sequence files. The files should be run through the analysis software that is supplied with your sequencer to provide initial processing or the raw data. For Applied Biosystems TM sequencers this will be the Sequencing Analysis Software.

5 Created by Damian Goodridge Page 5 of 38 1 Introduction 1.1 Overview Developed by scientists with extensive experience in DNA sequencing based HLA typing in a clinical HLA laboratory. Specifically designed for HLA SBT but applicable to any resequencing application including SNP scoring Removes data analysis as a bottleneck for high throughput HLA SBT / resequencing. User friendly, easy to use with minimum steps. 1.2 Unique Features Assign SBT TM 3 contains unique features for high throughput analysis. These features include: A base caller - for accurate base calling of heterozygous sequence. A base call scoring (BCS) algorithm - for the quality assessment of sequence base calls An intelligent algorithm for determination of the consensus sequence. A sequence alignment algorithm. A sequence matching algorithm. The simultaneous analysis of sequences for the resolution of heterozygous ambiguities eg codon 86 primers for HLA-DRB1 1.3 Summary of Functions Sequence analysis and allele assignment with Assign SBT TM 3 can be achieved in two steps. 1) Importing sequence files 100 s of sequences from multiple loci in a single step. Changing assays by sequencing additional exons does not require any changes to the software set up. Importing sequence files results in automatic: o Base calling and base call scoring.

6 Created by Damian Goodridge Page 6 of 38 o Reverse complementing of appropriate sequences. o Alignment with complementary strand. o Splicing of intron sequence. o Concatenation of sequence from multiple exons. o Calculation of consensus sequence and consensus BCS. o Identifies library for analysis. o Allele assignment by matching sequences within a library. 2) Confirmation of base calls / Sequence editing Rapid editing is facilitated by: Accurate base calling of homozygous and heterozygous sequence. BCS colour coded histograms allow rapid identification of regions of poor quality. Constant electropherogram viewing. Comparison of electropherograms between samples Priority analysis and editing of positions that are: o Low quality. o Mismatched against selected alleles from the results table. o Mismatched against all alleles in the results table.

7 Created by Damian Goodridge Page 7 of 38 2 Getting Started 2.1 Installation Assign SBT TM 3 is a single user application. It has been encrypted to prevent copying to other computers. You can obtain Assign SBT TM 3 as download from or on a CD. The file you will receive is called Setup.msi. Save this onto the hard disk of your computer. Once saved, double click on the Setup.msi icon and follow the installation instructions. During installation you will be asked to send you computer hardware number before being granted access to the software. The installer provides you with this number. Copy and Paste it into an message and it to conexio@iinet.net.au for licence registration.

8 Created by Damian Goodridge Page 8 of Operator Login Launching Assign SBT TM 3 by clicking the Assign.exe icon results in the opening of the Operator Login dialogue Select a registered user from the Operator: drop down list. Enter the password. The default admin password is cg01. This should be changed immediately by the administrator. Click Submit to login Changing Your Password or Default Settings To change the password or default settings: Select the required user Enter the current password Enter the new password in both fields Select the default reference Select the default encoding Adding New Users To add a new user:

9 Created by Damian Goodridge Page 9 of 38 Select the admin user Enter the admin password Enter a password for the new user Re-enter the password for the new user Enter the new users login name in the Edit Operators box Select the default reference Click Add User Encoding If you are using extended character sets please select an appropriate encoding system from the drop down menu. Encoding is currently available for Chinese (Big-5) and Japanese (Shift-JIS) characters. If you require additional support for this feature, please contact damian@conexio.iinet.net.au The Analysis Window Once logged in the following window appears. In order to perform sequence analysis using Assign-SBT TM v 3 the sequence file name convention and additional parameters must be defined in Edit Settings (See section 2.3)

10 Created by Damian Goodridge Page 10 of Setting the parameters for sequence analysis Select Edit Settings from the main menu The settings dialogue box allows you to: Create your sequence file naming convention Exclude primer site sequences where the primer site is within the exon. Customise your display including electropherogram colours Enter the location and sequence details of primers for the resolution of heterozygous ambiguities Select the library you wish to analyse according to your SBT protocol ie a homozygous library may be used for SBT protocols that separate alleles and result in homozygous sequences and a heterozygous library is selected for SBT protocols that do not separate alleles. Activate or deactivate automated editing of the consensus sequences.

11 Created by Damian Goodridge Page 11 of Settings Dialogue Box - Summary of Functions. The settings dialogue enables the operator to define the sequence file naming convention. The sequence filename must include the sample identifier, the locus and the sequencing primer. This is performed in the Sample Delimiters, Library Delimiters and Libraries panes within the Settings Dialogue. NB You must click on Update after making any changes to ensure that the alterations are recorded Sample Delimiters and Sample Naming Conventions Example 1: The Sample Delimiters pane enables the operator to define the location of the sample ID within the sequence file name. Eg if the sequence filename is A03_123456DRB1_F A03 = PCR well number = sample ID DRB1 = locus F = sequencing primer (Note: the PCR well number is optional. However, all sequence file names must contain the sample ID, the locus and the sequencing primer) The sample ID is located within the file name by either defining a unique character at the start of the sample name (in this case _ ) or by defining the start position within the sequence filename (in this case the 5 th position). The sample name length can also be indicated or a unique identifier can be used to indicate the end of the sample name. Example 2 If the PCR well number is not required in the sequence filename, a typical naming convention and appropriate settings are shown below The name for this sequence should be: SampleName_Locus_Primer.ab1

12 Created by Damian Goodridge Page 12 of 38 i.e _DRB1_F.ab1 If this convention is used the following can be entered into the settings window In this example a single character that defines the end of the sample name ( _ ) is used. (As values are required for Sample Delimiters Position, 0 is used). 2.4 Creating Locus Codes You have the option to define the codes you wish to use to identify the different loci within the sample identifier. A code must be identified for each locus even if the code you use is identical to the locus identifier in the Library Several codes can be used for the same locus. This is important for SBT strategies which employ allele group specific primers. For example: HLA B can be amplified with 2 primer sets based on the TA / CG dimorphism in intron 1. To discriminate between the primer sets the codes HLABTA and HLABCG may be used (or codes of your choice). Codes are set up in the Libraries pane area of the settings dialogue. In the drop down Locus: dialogue box select B (for HLA B). In the Code: dialogue box enter HBCG. Click on Add. Repeat to enter the code for HLA BTA. 2.5 The HLA Locus Prompt If Assign SBT TM 3 cannot find the locus code within the sequence filename the following prompt will appear as you attempt to import sequence files.

13 Created by Damian Goodridge Page 13 of 38 Select OK and the following dialogue will appear. Go the drop down menu and select the required locus. If the same locus is being typed for subsequent samples check the Use for subsequent samples: box. Select OK. 2.6 Primer Site Trimming Some SBT strategies (eg Atria Genetics HLA-DRB1) use amplification primer sites within exons. Sequence within these regions may be unreliable and of poor quality. This sequence can be removed using the Primer Site Trimming page. Select the Trim tab in the Settings dialogue.

14 Created by Damian Goodridge Page 14 of 38 Select the locus and the exon to be trimmed in the drop down menus. Enter the number of bases that should be excluded from the analysis. Click Update to record your changes. 2.7 Matching Mode Homozygous library mode is set for SBT protocols that separate the alleles by PCR prior to sequencing and result in homozygous sequence. Heterozygous library mode is set for SBT protocols that do not separate alleles and result in heterozygous sequence in the majority of examples (Note that the Heterozygous library mode can be used for homozygous protocols) 2.8 Library Mode Future versions of Assign will support the use of libraries such as FASTA files. 2.9 Heterozygous Ambiguity Resolution Primers (HARP) The Resolution dialogue enables the user to enter sequencing primer information used for the resolution of heterozygous ambiguities. Selecting the Resolution tab in the Settings Dialogue activates the resolution page. Please contact us for additional information regarding resolving heterozygous ambiguities by SBT

15 Created by Damian Goodridge Page 15 of 38 Use the drop down menu in Library to select the locus. Enter the start/end positions of the key nucleotides within the sequencing primer site which resolves the ambiguity (this could range from a single nucleotide to the entire primer). In the example above the 3 nucleotides in codon 86 (or nucleotides 343, 344 and 345) are entered. Enter the sequence between and including the start/end positions. In the example above, the HARP is complimentary to GTG at positions 343, 344 and 345. Select a name for the sequence primer e.g. codon 86. Select an alias (this a string that must be included in the electropherogram file name so that Assign SBT TM 3 can recognize the presence of the resolution primer). A typical file name would be _DRB1_GTG.ab1. When you have finished editing the information, select Update. See below section and section 5 for additional information regarding the use of HARP 2.10 Display Selecting Edit Display in the Settings Dialogue enables the colours of the electropherogram traces to be adjusted.

16 Created by Damian Goodridge Page 16 of 38 Change the color by selecting the base, choosing the colour and clicking on Set Colour Automated Editing This function uses information from the typing libraries to refine the base calling in the consensus sequence. A base call must meet a number of criteria before it can be reviewed by the automated editing function. 1. The Base Call Score of the consensus sequence at a site must be less than There must be signal present at the edited position for each base in the new call. 3. The algorithm is heavily weighted towards including extra bases rather than changing heterozygotes to homozygotes to minimise the possibility of incorrectly calling heterozygous positions. NB The automated editor will tend to favour base calls that are consistent with the libraries. As a result care should be taken to manually check areas where the sequence quality is poor. All automatically edited positions are stored in the edit list for a sample and highlighted in red above the consensus sequence. The edit list can be displayed by selecting the Edit List check box on the report generator. Any changes made by the automatic editor can be reversed by clicking on the Undo ( ) button 2.12 Updating the Settings Changes made to the settings must be saved by updating the Settings File

17 Created by Damian Goodridge Page 17 of 38 A number of settings files can be saved by entering a Settings File name for new parameters. Loading the appropriate Settings File will recover the settings parameters for subsequent analysis.

18 Created by Damian Goodridge Page 18 of 38 3 Sequence Editing and Allele Assignment 3.1 Importing sequences for analysis Go to File Import o Selected Sequences imports only the selected files. o Directory imports all the files in a given directory. You will be given the option whether you want to import sequences from sub-directories. Browse to locate the sequences to be analysed. Sequence files must be in.ab1,.abd, or.scf format. Data from many different loci can be analysed in the same analysis file. The sequences are displayed in the Analysis Window 3.2 The Analysis Window The analysis window is split into 3 panes. The first contains a list of samples, the second shows the electropherograms and sequences of the active sample, and the third contains a list of allele pairs that are best matched to the test sequence (See below).

19 Created by Damian Goodridge Page 19 of 38 Sample Pane Sequence Pane Assignment Pane The Test Sample Pane The first line contains the library name and date. Numbering is given by Exon and by Codon for the IMGT/HLA libraries. The active sample is highlighted dark blue with white text. Select the sample for analysis using the mouse, the arrow keys, or by dragging the scroll bar on the left. The test sample consensus sequence, the electropherogram (EPG) display and the allele assignments are updated automatically as you move between samples. Right clicking brings up the Sample Menu. Samples highlighted in orange have warnings associated with them. These warnings can be displayed by right clicking on the sample and selecting Show Warnings.

20 Created by Damian Goodridge Page 20 of The Sample Menu Selecting Auto Edit will launch the automatic editing process. This will review positions based on their BCS. Show Warnings lists any possible problems associated with analysis of the active sample. Add New Samples allows further electropherograms to be incorporated into a project. Remove Sample will erase the selected sample from the project. Remove All deletes all of the samples from the project. Confirmation will be required before completing this process The Sequence Pane The sequence pane displays information about the reference sequence, the consensus sequence and the electropherograms associated with the active sample. Scrollbar Mismatch Manual Edit Auto-Edit Confirmed Position Reference Consensus Consensus Base Call Scores Sample Consensus EPG Base Call Scores Electropherogram Sequence The Reference Consensus Editor Bar The top line contains the consensus of all the reference sequences in the library. This is the sequence to which sample sequences are aligned.

21 Created by Damian Goodridge Page 21 of 38 Each locus has its own reference sequence. The sequence positions begin at position 1 of the coding sequence as defined by the IMGT/HLA database and continue through all of the recorded exons Mismatch Positions All of the positions that represent mismatches against the alleles in the Assignment Pane are highlighted in yellow Confirmed Positions Positions that the operator has confirmed are highlighted in green. Please note that the confirmations are not saved in the analysis layout but are indicated on the report (See section 6) which can be saved Edited Positions Positions that the operator has edited are highlighted in blue. Positions that have been edited by the automated system are highlighted in red The Sample Consensus The sample consensus sequence and the base call score (BCS) histogram of the active sample is below the reference sequence. The consensus sequence is in a yellow shell for easy identification. The BCS histogram is colour coded. In Assign-SBT TM 3 the highest BCS (100) is white and lowest (0) is red. BCS between 1 and 100 are shaded from red to white. ( Note: a typical high quality consensus sequence from a single bi-directionally sequenced unit will have a BCS of approximately 90) Consensus sequence from positions where the automated editor has made a reassignment default to a BCS of zero. See Appendix 7.3 for the definition of the symbols used in the consensus. The sample consensus sequence is the only sequence which is edited and is the sequence on which allele assignment is performed. Editing is performed using the Editor Bar and Navigator (See section for a description of the Editor and section 4 for the description of the Navigator) Electropherograms (EPG s) Below the sample consensus sequence are the EPG s of each sequencing primer, the sequence base calls and the BSC histograms of the sequences.

22 Created by Damian Goodridge Page 22 of 38 Viewing the entire EPG is performed by dragging the EPG scroll bar to the left or right using the mouse. The sequence file name is indicated above each EPG. Zooming EPG is performed by pressing the Shift and one of the arrow keys. Up and down arrows adjust the vertical scale while the left and right arrows adjust the horizontal scale. Any of the individual traces can be hidden to facilitate analysis of heterozygous positions. Pressing Shift + A, C, G, or T will toggle the traces on and off. The trace intensity can be displayed by pressing Shift + I. Right clicking on a sequence will bring up the electropherogram menu. o Show Warnings will bring up a list of quality control warnings associated with the current sample. o Selecting Auto Edit launches the automated editing process. o Remove EPG will delete the active EPG from the sample. This is done when the sequence quality is poor compared to other EPG s for a sample and the poor quality sequence adversely affects the consensus base calling. o Trim Left removes all of the sequence to the left of the current position from the anlaysis. o Trim Right removes all of the sequence to the right of the current position from the analysis. NB Greyed out EPG is sequence that has extended into non-coding regions and has been spliced or regions selected to be trimmed by the operator. This sequence is not included in the analysis The Editor Bar The Editor Bar is a narrow, shaded box which extends vertically in the Sequence Pane. The reference sequence, consensus sequence and corresponding EPG sequences are aligned at the position of the editor. Consensus sequence at the position highlighted by the Editor Bar can be edited by pressing a key corresponding to an IUB code. Moving the Editor to view specific positions is performed by using the Navigator, the mouse or dragging the sequence scrollbar.

23 Created by Damian Goodridge Page 23 of The Assignment Pane Sample Name First Allele in the match Second Allele in the match Number of bases matched against both reference sequences Number of bases matched against one reference sequence Number of mismatches The assignment pane displays information about the best matched pairs of alleles from the current library. The selected test sample ID is indicated in the top left hand corner of the assignment pane. The results window contains the allele pairs whose combined sequence gives the closest matches to the observed sample sequence (columns Allele 1 and Allele 2). Homozygous samples contain the same allele name in each column. The number of mismatches between the allele pair combinations and the sample sequence are indicated in the MM columns. NB An allele assignment cannot be considered correct unless the number of mismatches indicated is 0 unless a novel allele is present. Alleles with up to 2 additional mismatches are also listed in the results window. Selecting an allele pair will result in the mismatched positions between the allele pair and the test sequence being listed in the Navigator. Selecting these positions in the Navigator enables the base calls at these positions to be verified. Editing incorrect base calls results in real time updating of the results table. NB Several allele pairs can simultaneously have 0 mismatches. This is because different heterozygous allele pairs may have identical sequence. These are heterozygous ambiguities. Some alleles are identical in the region sequenced. The Full and Part columns refer to the allele sequences in the reference library. They indicate whether both the allele sequences from the IMGT/HLA database for a given match have complete sequence coverage for the region being analysed.

24 Created by Damian Goodridge Page 24 of 38 0 in the Part column indicates that both library alleles have been completely sequenced. A number greater than 0 in the Part column indicates one of the alleles has not been completely sequenced over the region being analysed. The sum of Full and Part gives the number of consensus sequence bases used for allele assignment.

25 Created by Damian Goodridge Page 25 of 38 4 Sequence Editing Consensus sequence editing is performed using the Editor Bar and Navigator tool. The Navigator tool can be launched by selecting Launch Navigator from the menu or select N from the tool bar. 4.1 The Navigator Search Filters Sample Name Sample Selection Base Selection Layer Selection Confirm Base Call Current Position Information Search Filters Editing is performed on the consensus base, that is highlighted by the Editor Bar, using the base selection keys Current Position Information Indicates the Base Call Score, the current base call and the active position. In the example above the Base Call Score is 0, the base call is M and the Editor Bar is located at position 136. The drop down menu indicates the positions that are mismatched with the alleles selected in the assignment pane (not demonstrated in this example). Selecting positions in the drop down menu will take the Editor to these positions for sequence verification Search Filters

26 Created by Damian Goodridge Page 26 of 38 Checking the BCS and/or Mismatch boxes enables fast verification of bases with low Base Call Scores and/or consensus sequence positions which are mismatched with allele pairs within the assignment pane. When the BCS window is selected: Clicking the button will move the Editor Bar to the next consensus position with a Base Call Score of less than 30. Clicking the button will move the Editor Bar to the previous consensus position with a Base Call Score of less than 30. When the Mismatch window is selected, Clicking the button will move the Editor Bar to the next position that is mismatched with any of the alleles in the assignment pane. Clicking the button will move the Editor Bar to the previous position that is mismatched with any of the alleles in the assignment pane. When both BCS and Mismatch are selected the Editor Bar will be moved to consensus sequence positions where either the BCS is less than 30 or the base represents a mismatch with any of the allele combinations in the assignment pane Position Selection Button Function and Moves the Editor to the first and last base in the analysed sequence respectively. and Moves the Editor to positions that have low BCS or are mismatched with the allele pairs in the assignment pane (see 4.1.2) and Moves the Editor by a single base in either direction Sample Selection /\ Moves to the next sample \/ Moves to the previous sample Base Selection The base selection buttons are used to edit the sample consensus sequence.

27 Created by Damian Goodridge Page 27 of 38 Selecting a button switches a base at any position on or off to indicate presence or absence respectively. In the example above (in 4.1) the A, C and M buttons are shaded to reflect the M base call at this position. If M is an incorrect consensus base call and inspection of the EPG at this site confirms that A is present, the consensus sequence is changed by checking the C edit button. This will remove the C base call, leaving the consensus base call as A The consensus sequence and the assignment pane are updated automatically when consensus sequence positions are edited. The + button indicates a base insertion compared to the consensus sequence The button indicates a base deletion compared to the consensus sequence NB See Appendix 7.4 for more information regarding insertions and deletions The * button indicates that a base call at that position cannot be made. This position is excluded from the allele assignment. This button cannot be selected but shades darker when all other bases are deselected to indicate that a base call cannot be made at this position Layer Selection This function enables viewing of the heterozygous sequence (master) or the sequence generated by ambiguity resolving primers. Parallel editing of the two (or more) consensus sequences enables high resolution typing and resolving of heterozygous ambiguities. See below (Section 5)

28 Created by Damian Goodridge Page 28 of 38 5 Resolution of Heterozygous Ambiguities by DNA sequencing The ability to resolve heterozygous ambiguities by DNA sequencing is a novel feature of Assign-SBT TM 3. Heterozygous ambiguities arise when two or more allele combinations give rise to identical heterozygous sequence. The ambiguity arises because the cis-trans relationship between heterozygous base calls at various positions cannot be resolved. Identification and sequencing of one allele from the ambiguous combinations will conclusively demonstrate which pair of alleles is actually present. In order to achieve this, one of the alleles is amplified separately by using a sequencing primer specific to that allele. The original pair of sequencing primers and the heterozygous ambiguity resolving primer (HARP) can be imported together or the sequence from the ambiguity resolving primer can be included at a later date. The ARP information can be updated in the Settings dialogue by selecting the Resolution tab. An example of heterozygous ambiguity resolution is illustrated below. The locus is HLA-DRB1. The HARP is situated at codon 86. The results table contains an additional column MM 1, which contains the mismatches of the HARP sequence. MM 0 contains the mismatches of the PCR product sequencing primers (Master). MM 0 indicates that the test sequence has 0 mismatches with DRB1* , DRB1* , DRB1* and DRB1*

29 Created by Damian Goodridge Page 29 of 38 Selecting the 86 primer from the drop down menu on the Navigator displays the electropherograms, consensus sequence and mismatch data for the codon 86 HARP sequencing primer. The results table now highlights the alleles according to whether they are amplified by the ARP. The highlighted allele is amplified while the greyed out allele is not. For each heterozygous pair that contains DRB1* the MM 1 is 0 indicating that the sequence from the HARP is identical to HLA-DRB1* The correct match is the allele that has 0 mismatches in all the MM columns. In this case, DRB1* is the only combination that meets this condition. If neither allele is sequenced by the HARP, the MM 1 column will be filled with NA (not applicable) as in the example below.

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31 Created by Damian Goodridge Page 31 of 38 6 Reports Reports can be viewed, printed and saved following completion of sequence verification and editing. Select Reports Report Generator from the menu. 6.1 The Report Generator Report Type Output Selection Sort Order Output Filters BCS Analysis Selection Summary Reports Generate Quality Control Report Select Format Database submission - requires customisation Generate Create Report FASTA file

32 Created by Damian Goodridge Page 32 of Report Type There are currently three basic report types The allele report is used to display a detailed analysis of the match results. The table output sorts the results by sample name and locus. The allele summary gives a list of matches for each sample Output Selection The components displayed in the allele report can be adjusted as required. Match summary is a display of the allele pairs with the lowest number of mismatches against the observed sequence. NMDP codes can be displayed for each sample. The NMDP codes are calculated by generating match strings from the allele combinations that have the minimum number of mismatches against all the layers for the sample. The mismatch table shows all the allele combinations in the assignment pane, together with the mismatched bases for each of those combinations. The edit list contains all of the changes that have been made by the automated editing system and the operator Sort Order The output can be sorted by locus or by sample name Output Filters Reports can be filtered by sample name or by reference. In order to display a report for a single sample, click the populate sample box and choose the desired sample from the drop down menu. To filter the report by locus, choose the required locus from the locus list BCS Analysis Selection This is used to filter the bases that are included in the sequence quality analysis reports. The output can be restricted by exon, base position and sequencing direction BCS Report The Base Call Score Report shows the distribution of scores for the selected samples, together with the mean and standard deviations for each sample separately. This can be used to monitor sequencing performance FASTA file This produces file containing the consensus text sequence for all samples in standard FASTA format.

33 Created by Damian Goodridge Page 33 of Output Format Most of the reports can be generated as a text file, or incorporated into an Excel spreadsheet. The exceptions are the FASTA file, which can only be produced as a text file and the BCS report, which is only available in Excel form Database submission Submission to a database requires customised modifications to the Assign SBT code. Please contact damian@conexio.iinet.net.au for further information regarding this feature Generate Report Clicking this button creates a report with the selected characteristics.

34 Created by Damian Goodridge Page 34 of 38 7 Saving / Closing / Opening On completion of analysis: select File Save to save a project. To re-open a saved file After logging in go to File Open. Browse to the saved file to open it. NB Assign SBT TM 3 does not update the electropherogram files. In order to re-open a project, the original electropherograms must be kept in the same location after closing the project.

35 Created by Damian Goodridge Page 35 of 38 8 Appendix 8.1 Menu Functions Menu Function File New Creates a new project Open Opens an existing Analysis File Save Saves a working Analysis File Save As Saves a working Analysis File under a new file name Import Selected files Import selected sequences Directory Import all sequences in a directory Print Not available in this version Print Preview Not available in this version Print Setup Not available in this version Exit Close / Exit Assign SBT TM 3 Edit Undo Undoes base call edits Find Launch the search dialogue Settings Allows the operators to enter their sequence file name conventions and to select the analysis range for loci where amplification primers are located in exons View Tool bar Removes or includes the tool bar in the Analysis Window Status bar Removes or includes the status bar in the Analysis Window Launch Navigator Launches the Navigator Tool Reports Report Generator Launches the Report Generator dialogue Help About Assign Assign SBT TM version 8.2 Sequence Libraries and Assign SBT TM 3 Applications Sequence libraries have been generated for MHC coding sequences provided by the IMGT/HLA database. The sequences are located at NB Although Assign SBT TM 3 can be used for SBT of any loci in the library, only HLA A, B, Cw, DRB1, DRB3,4,5, DPB1, DQB1, DQA1 have been tested.

36 Created by Damian Goodridge Page 36 of 38 The standard library list for version 3 is: HLA A HLA B HLA Cw DRB1 DRB3,4,5 DPB1 DQB1 DQA1 DMA DMB DOA DOB DPA1 DRA HLA E HLA G MICA TAP1 TAP2 8.3 Consensus sequence symbol definitions * = No base call. Positions assigned as * will not be processed by the assignment algorithm. This symbol can be used to replace base calls if the operator is uncertain of the sequence. A = Adenine C = Cytosine G = Guanine T = Thymidine R = A+G D = A+G+T Y = C+T H = A+C+T K = G+T V = A+C+G M = A+C N = A+C+G+T S = C+G W = A+T B = C+G+T + = inserted sequence compared to the reference.

37 Created by Damian Goodridge Page 37 of 38 = deleted sequence compared to the reference. Lower case letters denote positions where one sequence making up the consensus contains a deletion and another contains a valid base. 8.4 How Assign SBT TM 3 Handles Insertions and Deletions Insertion Notification The Assign SBT TM 3 alignment algorithm uses a single symbol in either the consensus sequence or the sequences which contribute to the consensus ( + for insertions and for deletions) to indicate insertions or deletions relative to the reference sequence. The number of bases inserted, together with the insertion sequence, is displayed in the Insertion Notification box of the navigator. In the example above the following sequences occur immediately left of the editor Upper electropherogram sequence: RCAAGT Lower electropherogram sequence: RCAAAGT

38 Created by Damian Goodridge Page 38 of 38 In order to maintain the alignment a (+) has been used to indicate the insertion. This is carried through to the consensus sequence unless a filter has been applied. Please note that the inserted bases occur before the highlighted position. The BCS at this position is set to 0. All positions with a + or in the consensus have a default BCS of 0. This is to ensure that whenbcs is selected as the fast track editor option, the insertion/deletion base calls will not be missed. In addition, when Insertions and Deletions are DESELECTED in the Assignment windows of the Settings dialogue box, these positions will register as mismatches with the allele library. 8.5 Contact Us: We are always grateful for any suggestions or comments regarding the software. David Sayer Damian Goodridge Conexio Genomics Conexio Genomics PO Box 1670 PO Box 1670 Applecross 6953 Applecross 6953 Western Australia Western Australia T: +61 (0) T: +61 (0) david@conexio.iinet.net.au damian@conexio.iinet.net.au