Surveyor. DNA Variant Analysis Software. Mutation. SoftGenetics LLC. v Innovation Blvd, Suite 235 State College PA USA 814/237/9340

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1 Mutation Surveyor DNA Variant Analysis Software v 3.1 SoftGenetics LLC 200 Innovation Blvd, Suite 235 State College PA USA 814/237/ info@softgenetics.com technical service: tech_support@softgenetics.com copyright 2007

2 Table of Contents TABLE OF CONTENTS TABLE OF CONTENTS...1 CHAPTER 1 INSTALLING MUTATION SURVEYOR... 3 SYSTEM REQUIREMENTS... 4 LOADING THE SOFTWARE... 4 REGISTRATION... 5 CHAPTER 2 FEATURES OVERVIEW... 6 SUMMARY...7 NAVIGATION... 7 MAIN MENU OPTIONS... 8 MAIN TOOLBAR ICONS... 9 COLOR CODES AMINO ACID CODES CHAPTER 3 GENERAL PROCEDURE UPLOAD FILES One Directional Two Directional DATA WINDOWS File Tree ORT GAD...22 MUTATION CALLING PARAMETERS WHAT TO EXPECT MUTATION VERIFICATION CHAPTER 4 OUTPUT REPORT TABLE (ORT) OVERVIEW Output Report Table Nomenclature General Functions MAIN TOOLBAR OPTIONS Saving the ORT Display 1D / 2D Settings Two Directional ORT Advanced Two Directional ORT HGVS ORT Graphic Display of Mutations Customer Reports Nucleotide Text Output DNA Quantification CHAPTER 5 GRAPHIC ANALYSIS DISPLAY (GAD) OVERVIEW Graphic Analysis Display General Functions WHAT TO EXPECT MAIN TOOLBAR OPTIONS Detect Heterozygous Indels

3 Table of Contents Methylation Detection CHAPTER 6 FILE EDITORS FILE TYPES Trace Files GenBank Files FILE FORMAT CONVERSION SCF from SEQ/GBK SCF from AB1/ABI/FASTA SCF/SEQ/GBK from SGP EDITORS File Name Editor SEQ File Editor GBK File Editor Advanced GBK File Editor Log File Editor and MutAutoRun Print Header Editor Filename Match Editors ADDITIONAL TOOLS SGP Comparison Mutation Assembly Mask Vector Sequence Sequence Assembler Export Trace Data CHAPTER 7 REPORTS, SAVING AND PRINTING REPORTS Report Types Clinical Report SAVING Save Files Save Project Save Report Table Save Base-Modified Samples Save Pictures as a Word Document PRINTING Data Tables Graphic Displays FREQUENTLY ASKED QUESTIONS INDEX

4 Chapter 1 Installing Mutation Surveyor Chapter 1 Installing Mutation Surveyor Mutation Surveyor... A Unique Research Tool Mutation Surveyor is available in 400 and 48 lane capacities. Utilizing our patented anticorrelation technology, the program rapidly locates all differences between the wild type sequence and sample traces with excellent accuracy and sensitivity. The program can be used with either single direction or bi-directional data. Before installing Mutation Surveyor/Explorer, please review the system requirements that are listed in this chapter. NOTE: For the remainder of this manual, the program name Mutation Surveyor should be understood to be interchangeable with Mutation Explorer except where specifically noted. Chapter 1 Contents System Requirements Loading the Software Registration 3

5 Chapter 1 Installing Mutation Surveyor System Requirements In order to load the software into your computer, you will need the following: Pentium III processor with 1.8 GHz or higher CPU with 512 MB or higher Hard drive with at least 10 GB available space Microsoft Windows 2000, NT or XP (Network version requires server with 32 bit Windows XP CD-ROM drive Operating key (not needed for Evaluation or Demo versions) Mutation Surveyor/Explorer CD Operating Key The operating key is a small piece of hardware that came in your order. Since there are several versions available (depending on your computer hardware), each one has specific instructions included with the key. You must attach this key to either a USB or a parallel port in order for the software to recognize your machine as a licensed user. Windows operating system NT requires use of parallel port key, as USB ports are not typically recognized by NT. If you received a USB key and have Windows NT, please contact SoftGenetics for an exchange of operating keys. Once you have installed the operating key, it is best to not remove it. If you do remove the operating key from your machine you will need to re-set your machine to accept this piece of hardware. Loading the Software 1. To Install Mutation Surveyor, You are required to have administrative privileges. 2. Load the CD into CD-ROM drive, contents of CD should appear on the opening screen. If contents do not appear, open My Computer and select the CD drive. 3. Double click on Mutation Surveyor Setup.exe or Mutation Explorer Setup.exe 4. Follow the steps for the Installation Wizard. The default will load the software to C:\Program Files\Mutation Surveyor OR Mutation Explorer. 5. Insert the operating key into the appropriate port. A Found New Hardware message will appear. 6. Click Yes to install the drivers. 7. IF a Hardware Installation warning box appears warning you that this device is not Microsoft XP approved, click Continue Anyway and then Finish on the following box. 4

6 Chapter 1 Installing Mutation Surveyor Registration When you open the software for the first time, you will be prompted with a dialog box to register the product. Users have one of two registration modes to choose from, either Online Registration or Offline Registration. Each software key has a unique User ID that will be automatically loaded into the User ID box when either online or offline registration is selected. Please locate the Account and Password on the software CD you received. You will need your Account and Password to register your copy of the program. Online Registration (Users with Internet access) 1. Select Online Registration in the dialog box. 2. Click Next. 3. Enter your address, account and password (printed on the CD) into the corresponding fields. 4. Click Register. Your software will be registered automatically. A confirmation will be sent to you as well. 5. Re-launch the program. Offline Registration (Users without Internet access) 1. Select Offline Registration in the dialog box. 2. Click Next. 3. Copy and paste the User ID, enter the account and password (printed on the CD) into the body of an and mail to tech_support@softgenetics.com. 4. The Registration ID will be sent to you within one business day. 5. Copy and paste the Registration ID from the into the Registration ID field. 6. Click OK. 7. Re-launch the program. 5

7 Chapter 2 Features Overview Chapter 2 Features Overview Mutation Surveyor and Mutation Explorer utilize our patented physical trace comparison technology providing accuracy >99% when using bi-directional Phred 20 quality sequence traces. The software is an excellent tool for both discovery and diagnostic applications, and accepts sequence traces from all major sequencing systems such as those marketed by Applied BioSystems, Foster City CA, or Amersham Megabace systems, and is compatible with either terminator or primer chemistries. The software s unique technology provides the best tool for locating all DNA variants: homozygous and heterozygous single nucleotide polymorphisms and homozygous and heterozygous insertions or deletions (indels). Both programs will automatically deconvolute hetero-indels into two clean traces for rapid and accurate analysis of insertions and deletions from 1 to 200 base pairs. To be user-friendly software, Mutation Surveyor was designed to follow standard Microsoft platform conventions established in software packages such as Word and Excel. Chapter 2 Contents Summary Navigation Main Menu Options Main Toolbar Icons Color Codes Amino Acid Codes 6

8 Chapter 2 Features Overview Summary The program is broken up into two analysis modes: the Output Report Table (ORT) and the Graphical Analysis Display (GAD). The analysis modes present the location and characteristics of each mutation in either textual or visual formats, respectively. Moving within the ORT and GAD windows is primarily icon-driven and follows standard Microsoft Windows conventions. Switching between these two analysis modes is done by: ORT to GAD Double-click on any mutation or sample name to go to the correct mutation location in the GAD for that sample. GAD to ORT Click on the Run icon OR Display 1Dir Output OR Double-click Mutation Report in the File Tree frame. GAD ORT Right-mouse click In different locations throughout Mutation Surveyor the right-mouse click action will activate action menus. These items are also described in the relevant locations in this manual (See Chapter 4 ORT and Chapter 5 GAD). Navigation Zoom in Hold down left-mouse click and draw a box from the upper left corner of the window towards the lower right corner. Zoom out Hold down left-mouse click and draw a box from the lower right corner of the window towards the upper left corner. Scroll Hold down the right-mouse key and drag the trace in either horizontal direction. Move Between Peaks Use Ctrl+F to move to the next mutation peak. Use Ctrl+B to move to the previous mutation peak. 7

9 Chapter 2 Features Overview Main Menu Options File New Project Selecting New Project will close current project and prompt user to Save. Close Project Select Close Project to properly terminate project analysis prior to exiting. Open Files Select Open Files to upload reference and sample files. Open/Save Mutation Project (*.sgp) The entire Project file can be saved and reopened for later analysis. Open/Save/SaveAs File Link (*.prj) Saves and reopens only the file path to the sequence data, therefore *.prj files cannot be reopened on a separate computer. Open/Save Mutation Report Saves the ORT in.txt file format for later analysis Open Whole Gene Data Opens Whole Gene *.SGP Files for comparison with GenBank sequences or Mutation Report saved as.txt Exit Closes the program Process Run From ORT, Run analyzes raw sequence data. From GAD, Run toggles back to the ORT Restart Refreshes analysis settings. Select Restart after changing parameter settings or after selecting a new reference file Options Opens Options box for setting parameters. See Chapter 3 General Procedure Mutation Calling Parameters for further explanation of parameter features Display Frame Toggles Opens and closes frames in the main analysis window. 1Dir Output Displays the One Directional Output Report Table (ORT). 2Dir Output Opens Two Direction Output (2D ORT) window. 2Dir Advanced Opens Two Direction Output window with Advanced Settings box (Advanced 2D ORT). Graphic Display Opens the Graphic Display window. Print Display Opens the Print Display window. 8

10 Chapter 2 Features Overview Tools Editors Individual File Editors are discussed in detail in Chapter 6 File Editors. Additional Tools SGP Comparison window compares two separately analyzed project files for identification of differences. Mutation Assembly allows the user to upload several project files and display only sequences in those files with mutations. Mask Vector Sequence allows software to hide mutations detected in regions of vector DNA. Sequence Assembler assembles short sequences into one text string. Output Trace Data enables the generation of a text file containing bases and intensities. These tools are discussed in detail in Chapter 6. Help Help Launches the Mutation Surveyor manual. Hot Keys Opens box with quick navigation hits. Update Contact Online When selected, it updates the SoftGenetics database with your registration information. SoftGenetics Homepage Launches the SoftGenetics Homepage in your internet browser. About Opens an information box which indicates the date and version of the software package. Main Toolbar Icons This list is designed to be used as a reference tool while you learn those icons unique to Mutation Surveyor. For each icon, the name given is that which appears when your cursor is above that icon. The first page reference directs you to the first time this icon is introduced; this context will help you understand their specific functions. In the locations cited here, the icon can be found in the right margin. ORT Icons Open Files Opens File Upload box Raw Data to Processed Data Activates when *.abi files without base call information are uploaded. Converts the files to display base call information. Run Analyzes data Restart Refreshes parameter settings Show/Hide Browser 9

11 Chapter 2 Features Overview Toggles the File Tree window Save Saves the Mutation Report (.txt,.xls,.xml,.htm) Select Reference File Opens Contig Files box for reference file selection (not available in reopened Project (.sgp) file) Paired Two Direction ORT Opens Paired 2D Output window HGVS/Advanced Two Direction ORT Opens additional Mutation Score Settings box Graphic Display of Mutations Opens Graphic Display window Display mutations with 1D/2D settings Toggles between 1D/2D settings Customer Output Displays customer requested reports Show Nucleotide Text Shows contigs in text format Save Base Modified Samples Opens file tree to save modified sample files GAD Icons Reprocess Modified Files Appears when a base has been changed in a Sample file Quantify Opens DNA Quantification table Open Files Opens File Upload box Raw Data to Processed Data Activates when *.abi files without base call information are uploaded. Converts the files to display base call information. Run Analyzes data Restart Refreshes parameter settings Show/Hide Browser 10

12 Chapter 2 Features Overview Toggles the File Tree frame Show/Hide Nucleotides Toggles the Nucleotide frame Show/Hide Amino Acids Toggles the Amino Acid frame Show/Hide Peak Table Toggles the Mutation Peak Table Zoom In Zooms in on the center of the window Zoom Out Zooms out on the center of the window Show Lines Provides the option to Hide All/Show All or select specific nucleotides Select Reference File Opens Contig Files box for reference file selection Two Direction Output Opens the Two Direction Output analysis window HGVS Output Opens the Advanced Two Direction Output settings box Graphic Display of Mutations Opens Graphic Display window Print Display Opens the Print Display window Print Clinical Report Opens the print preview window of the Clinical Report Display mutations with 1D/2D settings Toggles between 1D/2D settings Detect Heterozygous Indels Opens Heterozygous Indel Detection analysis window Customer Output Displays customer requested reports Show Nucleotide Text Shows contigs in text format Save Base-Modified Samples Opens file tree to save modified sample files 11

13 Chapter 2 Features Overview Reprocess Modified Files Appears when a base has been changed in a Sample file Save Pictures as a Word Document Exports the Mutation Report as a Word (.doc) file with a trace file graphic Quantify Opens DNA Quantification table Additional Icons Two Direction Output Copy Sends current analysis view to the clipboard for paste into other programs (such as Excel and Word) Save Saves the current analysis as a Text (.txt) file Advanced Two Direction Output Opens Advanced Two Direction Output settings box Whole Gene Output Opens Whole Gene Output analysis box with options for sequence comparison Output Settings Opens Display Options box to adjust display settings JHU Output Opens customer specific analysis report window Tassel Output Opens customer specific analysis report window Emory Output Opens customer specific analysis report window Compare Mutation Output for Different SGP Opens file tree box to select a previous Mutation Report (.txt) file for comparison with the current project file Graphic Display Advanced 2D Graphic Display Opens Advanced Settings box to change display options Whole Gene Analysis 12

14 Chapter 2 Features Overview Opens Whole Gene Settings box with display options and Filename Assembly Options Print Clinical Report Zoom to Fit Allows user to view full Report in one screen Zoom to 100% Zooms Report to 100% aspect Zoom to Width Maximum zoom in on Report Printer Setup Opens Page Setup options box Print Select Printer and print Heterozygous Indel Detection Auto Identify the Start Automatically initiates a secondary search of traces for a possible alternative start point for the indel. Quantification Auto Create Groups Opens DNA Quantify options box with data processing options Edit Groups Opens Quantification Group Editor window for each Contig Options Opens DNA Quantify Options box with report filter and columns options SGP Comparison Load SGP Files Loads SoftGenetics Project (.sgp) files for comparison Put the Files into Different Groups Opens Filename Assembly Settings box Select Standard Allows user to select a project file for use as the standard settings file Compare Mutation Output for Different SGP 13

15 Chapter 2 Features Overview Compares mutations in each project file and identifies differences between the mutations Whole Gene Output Whole Gene Sample Assembly Opens Sample Assembly window to define filename substring Consensus Sequence Text Opens Sequence Text Output window to group sequences in a specific range and displays in text format Sequence Text Comparison Opens Sequence Text window and displays sequence differences in text format Amino Acid Text Comparison Opens Amino Acid Text Output window and displays sequence amino acids in text format Homolog Consensus Sequence Text Displays Sequence text from the Advanced Two Directional Output window Sequence Text Output Display Sequence in One Line Toggles between displaying the sequence in one line or in paragraph form Save as PHD Files Opens file tree to save sequence text as a.phd file Color Codes JHU Output Display the Sequence of ROI Opens Text Sequence window to view text sequence in Region of Interest File Tree Selected files in the file navigation window are highlighted in green, unselected files are standard black. ORT Blue Text high confidence that it is a mutation Red Text low confidence that it is a mutation Black Text mutation has been confirmed Purple Background the mutation has been recorded in GenBank Pink Background the mutation resulted in an amino 14

16 Chapter 2 Features Overview acid change (missense and nonsense mutations) No Background Color the mutation did not result in an amino acid change (silent mutation) 2D Paired ORT Blue and Yellow Background is used to visually highlight matched sample pairs. Purple Background the mutation has been recorded in GenBank Pink Background the mutation resulted in an amino acid change (missense and nonsense mutations) No Background Color (n.a.) Called mutations have been filtered by userdefined parameters. GAD Electropherogram Trace Colors There are a total of 16 possible colors - 4 are the standard colors of the 4 main bases while the remaining 12 are for the heterozygous pairings. These colors are selected via the Show Lines icon in the main toolbar. Sequence Text are color-coded according to this same scheme. Light Tan bar - GenBank sequence match Yellow arrow bar - Exon-coding region Pink bar Region of Interest (ROI) Light Blue bar Primer regions Dark Red bar Insertion/Deletion Short Red bar - Duplication Short Green bar Somatic (Mosaic) Mutation (2D small peak) NOTE: Parameter values associated with mutation peaks are color coded using the same scheme as is used for mutation font coloring in the ORT. Processable data is bracketed by blue vertical bars at the beginning and the end of mutation electropherogram. The blue bars also indicate the Vector trim boundaries, if you select to use the vector trimming option. Potential mutation identification In an electropherogram trace, the software will add a red dot at the peak of the nucleotide if the relative intensity drops 35% but does meet the other requirements of the mutation score. This indicates a potential mutation; further examination will then be required to determine if this mutation is real or a chemical artifact. Sequence Text Frame Colors Direct sequence notation The topmost panel of the GAD is the direct base calling text for the reference, sample and GenBank-derived sequences. Below the bases a black star (*) indicates an inconsistency of the base calling between the reference file and the sample 15

17 Chapter 2 Features Overview file, while a red star (*) indicates an inconsistency between the reference file and the GenBank file. Mutation Data Table Colors Confirmed mutations are changed to black text in the mutation data table at the bottom of the GAD. Unconfirmed mutations are in blue or red text. Other colors are discussed above in the ORT section. Amino Acid Codes 1st and 2nd Base 3rd Base T C A G TT Phe F Phe F Leu L Leu L TC Ser S Ser S Ser S Ser S TA Tyr Y Tyr Y Stop Stop TG Cys C Cys C Stop Trp W CT Leu L Leu L Leu L Leu L CC Pro P Pro P Pro P Pro P CA His H His H Gln Q Gln Q CG Arg R Arg R Arg R Arg R AT Lle I Lie I Lle I Met M start AC Thr T Thr T Thr T Thr T AA Asn N Asn N Lys K Lys K AG Ser S Ser S Arg R Arg R GT Val V Val V Val V Val V GC Ala A Ala A Ala A Ala A GA Asp D Asp D Glu E Glu E GG Gly G Gly G Gly G Gly G 16

18 Chapter 3 General Procedure Chapter 3 General Procedure Researchers no longer need to perform time consuming and inaccurate comparison of the entire sample trace. With Mutation Surveyor, any found variants of the sample when compared to the wild type are clearly indicated in our mutation electropherogram as a sharp peak. Accuracy of the software in the bi-directional analysis mode is over 99%, with sensitivity to greater than 5% of the primary peak. Our collaborators have demonstrated an accuracy of 95% when processing single direction sequence traces. Mutation Surveyor s detection sensitivity has been enhanced to report similar peaks that are buried in baseline noise of both the forward and reverse traces, alerting researchers to the possible presence of mutations buried in the background noise. Mutation Surveyor easily processes 400 lanes of data in approximately two minutes and can be operated on a fully automated unattended basis. The software automatically forms contigs, performs alignments and mutation detection comparing both forward and reverse patient traces to reference or normal traces. Homozygous and heterozygous mutations are indicated by sharp peaks in our exclusive mutation electropherogram. Chapter 3 Contents Upload Files One Directional Two Directional Data Windows File Tree ORT GAD Mutation Calling Parameters What to Expect Mutation Verification 17

19 Chapter 3 General Procedure Upload Files Entering Files - General 1. Open the software. You will see a graphics page and a simple toolbar. 2. Click on the Open Files icon. The Open Files window will appear. 3. Use Add (standard Shift and Control commands allow you to add multiple files at once) to select GenBank, reference and sample files. Click Open to enter them into the file entry window and then click OK. a. For One Directional data (1D), enter a single reference and up to 400 samples, all oriented in the same direction. b. For Two Directional data (2D), enter a forward and reverse reference as well as a matched set of samples. File names that differ only with a directional (F or R) will be automatically grouped properly. NOTE: Up to 400 samples (or 200 pairs) can be entered into a single analysis session. For tools to process larger numbers of samples, see MutAutoRun in Chapter 6 File Editors. 4. Select Process Options from the main toolbar. In the Options box, click Default and then click OK. (See Mutation Calling Parameters section for more information) 5. Click the Run icon to run the mutation analysis. Files will be aligned into contigs and presented in the File Tree frame on the right of the main analysis window. NOTE: Reference and sample traces are limited to 1200 base pair read lengths; GenBank sequences do not have this limitation. This is generally not a concern since resolution of most systems is much less than 1200 bases. File Types Sample file the unknown to be Explored or Surveyed in SCF, AB1/ABI or Gz format Reference file a control either wild type or with known characteristics used to compare the samples with can be either user-entered, lab-derived OR created from a GenBank file in SCF, AB1/ABI or Gz format GenBank file can serve as a reference available on the internet will generate a trace either from base calls alone or with lab-generated data as well in SEQ or GBK format provides amino acid identification and changes within the coding region, nomenclature, and allows the transition between genomic and cdna 18

20 Chapter 3 General Procedure Entering One Directional Files 1. Identify your files. 2. Open the software. You will see a graphics page and a simple toolbar. 3. Click on the Open Files icon 4. The Open Files window will appear. Click Add to enter individual sequences, or groups of sequences into each of the sections. 5. If you wish to compare all of your samples against each other, simply enter all the samples into the reference box, click on Add R S. This will add the same group of traces into the sample dialog box. NOTE: Files may be entered as a number of file types; using *.seq is the ideal. A full discussion of file types and file conversions can be found in Chapter 6 File Editors. 6. After adding files, click OK. The File Tree will open on the right side of the main analysis window. Contig folders can be expanded to show specific files. Doubleclicking on a file will call up the raw electropherogram for that sample. 7. To run the analysis, click the Run icon in the main toolbar. Automatic GenBank Upload When analyzing human DNA, the user has the option to leave the GenBank and Reference File fields empty. The software will automatically access the GenBank database and search for the correct gene to match the sample files. Using a GenBank-derived file as a reference ensures that one will have the correct base count, amino acids will be included in the coding region and previously reported mutations will be included and identified in the analysis. Mutation Surveyor will create a modulated reference trace from a GenBank-derived file if either the GenBank-derived file is entered into the Reference window or if the Reference field is left empty and a GenBank-derived file has been entered. While using a GenBank-derived file as the reference may introduce some false readings, most users have found that when using high quality sequence samples, it is an excellent reference and that the added benefits far outweigh the introduction of a few false readings (see Chapter 6 File Editors). Raw Data Window Entering Two Directional Files This option will quickly become standard for most users because the dual-direction analysis is much more powerful than a simple, 1D mutation identification. There are 3 ways to enter 2D data directly with Loose Match, directly with Exact Match or with the automatic Load 2D Match tool. Which one you choose will depend on the naming convention you use (See Naming Conventions below). NOTE: No matter which convention you use, you will need to enter 2 reference files one for the forward and one for the reverse direction. 19

21 Chapter 3 General Procedure Naming Conventions It is best to follow a strict convention when performing analysis of forward and reverse sequence pairs by including _f_ in the name of the Forward Sequence and _r_ for the Reverse Sequence. The software will then automatically pair the sets, if there is the same number of samples in each direction. NOTE: Files with similar filenames (except for F/R) will be considered as from the same sample or patient. If your filenames do not follow this kind of convention or have extra information that makes it difficult to pair files, use either Load 2D Match or Filename Editor (see below). Adding Files using Loose Match This is the default setting. In this mode Mutation Surveyor is able to see that these files are very similar and that one has an F and one has an R in the paired filenames; and then decides that they should be paired. This is a good option when your filenames have information like capillary number or plate number. EXAMPLE: MMipR260_f_017.ab1 / MMipR260_r_018.ab1 These files will be read as a forward/reverse pair even though there are two differences between the filenames: f/r and 017/018. Adding Files using Exact Match If you want to be more cautious and your paired filenames are exactly the same except for an F or R to indicate the forward and reverse complements, then you can use this setting. To change from Loose Match to Exact Match, you need to check the Exact Filename F/R Match button. To do this select Process Options in the main toolbar and click the Contig Options tab. EXAMPLE: MMiOpR260_f_018.ab1 / MMiOpR260_r_018.ab1 In this example, only the f/r is different. Pairing text files using the Load 2D Match tool For some users, neither of the above entry styles is ideal. For example, your naming convention may require extensive renaming in order to use one of the automatic pairing methods (Loose Match or Exact Match). Additionally, creating text files entered with the Load 2D Match tool may be an efficient way to set aside blocks of data to be analyzed at a later time or by different users. 1. Convert the sample files to Text (.txt) format. This can be done by one of two ways - with the 2D Filename Match Editor (Chapter 6 File Editors) or with Microsoft Excel. NOTE: When creating the Text (.txt) file, all samples in the forward direction must be in one column and all samples in the reverse direction must be in a separate column. Each row in the matrix represents one sample, 20

22 Chapter 3 General Procedure therefore all the files in the same row will be considered from the same sample. 2. Click the Open Files icon to launch the Open Files window. 3. Check the Load 2D Match option at the bottom of the window. 4. Click the Add button next to the Sample Files field and select the Text (.txt) file you just created. Important Notes: The text file that contains your filenames list MUST be stored in the same directory as the trace files. If they are stored in a different directory, you will need to include that information as part of the trace name. In most cases, users have one reference trace and multiple samples. To use Load 2D Match tool in this instance, you will need to create two separate text files - enter one text file into the reference window and one into the sample window. Do not add the trace files the program will automatically enter them according to the Excel or text file. Multiple Primer Pairs If multiple primer sets are used, Mutation Surveyor can sort them into a single sample from a spreadsheet program like Excel. In order for this tool to work properly, use the ND Filename Match Editor tool (Chapter 6 File Editors). NOTE: Grouped filenames must be in the same row. Data Windows Contigs and File Tree Window Alignment of GenBank-derived, reference and sample files is automatically performed by the Contig calculation. The sorting parameters used for this operation are fragment size, minimum matching base number and minimum matching base percentage. These settings may be adjusted in the Options window (Chapter 3 General Procedure - Mutation Calling Parameters). Files with similar sequences of base call texts, with matching user-specified parameters, will be grouped into a contig. These files will then be aligned with the sequence text. Files will be displayed in the File Tree frame of the main analysis window. NOTE: The number in parenthesis after the contig name indicates the number of samples in that contig. Since contigs are organized relative to the reference file, forward and reverse sequence pairs are sorted into two contigs and labeled as contig xf and contig xr (where x stands for a specific filename). While forward and reverse samples are grouped separately, they are displayed together. Green highlight indicates that a file is active. Red check indicates the active reference file. Black check indicates a GenBank-derived reference file. 21

23 Chapter 3 General Procedure File Navigation Expanding folders Folders can be opened or closed (also referred to as expanded and shrunk) by clicking on the [+] or [-] sign. Calling up raw traces Expand the folder labeled File. Double-click on an individual filename; the raw electropherogram for that file will be displayed. This can be done for all three file types (GenBank, reference and sample) simultaneously, for a maximum of three traces being displayed at one time. Changing reference file To change the reference file to any other file in the contig, click the Select Reference File icon. This activates a window with all the files. Hold the CTRL key and select/deselect the desired files. Click OK; the reference file in that contig will be replaced and the mutation electropherogram will be recalculated. Output Report Table (ORT) Presented in table format is the following information: Number Sample File Name Reference File Name Dir notes the directions to the GenBank file. Gene Name a note taken from a *.seq or *.gbk file. Exon Name a note taken from a *.seq or *.gbk file. Reading Frame the reading frame number of the first base in an exon. Start Base # the number of the base at the start of processable data (start blue bar). End Base # the number of the base at the end of processable data (end blue bar). Lane Quality is the measure of the physical quality of the reference and sample traces. Mutation Number the number of mutations found in this sample sequence. The mutation number -1 means the quality is low. Mutation Code is the abbreviated name that is unique for each mutation in a sample and presents: base number, reference nucleotide, mutation nucleotide and mutation score. NOTE: See Chapter 4 ORT for features of this analysis mode. Graphic Analysis Display (GAD) The GAD window includes the following panels: Direct sequence base calls with corresponding amino acids. Sequence traces for both reference and sample normalized to average peak intensity and presented on a scale suitable for maximum visual impact. Mutation electropherogram displays the found differences between the reference trace and the sample trace, based on the parameters outlined below. Mutation data table summarizes all the mutations found for the selected sample, presenting location, score and defining values for mutation score. NOTE: See Chapter 5 GAD for features of this analysis mode. Two Directional Analyses 2D data is presented slightly differently in both the ORT and the GAD. Briefly, the Two Directional Analyses mode accommodates the more complex grouping of data and the increased number of traces relevant to each sample. Mutation Calling Parameters Mutation Surveyor physically compares a sample trace to a reference trace and identifies found differences based on the satisfaction of user-defined parameters. If ALL 22

24 Chapter 3 General Procedure parameters are satisfied, then the differences are indicated as a potential mutation, with a confidence call provided. As well as the parameters discussed below, peak front, base peak labeling and baseline removal are taken into consideration. Functionally, this process mimics what researchers have been doing visually for some time and is much more stringent than simply comparing base call text. NOTE: Mutation Calling Parameters cannot be changed in Mutation Explorer the default settings are fixed. The following Options window can be accessed by selecting Options from the Process menu in the main toolbar. Default settings are in parentheses. Raw This window allows the user to choose which type of *abi file was uploaded. Load Raw Data Raw Data refers to *abi files that do not contain base call information (ATCG). If these type of *abi files were uploaded, select Load Raw Data so Mutation Surveyor will extract base call information from the files. Load Processed Data (Selected) is the default setting because most trace files uploaded will include base call information. Contig The elements in this window impact how Contigs are assembled from sample files. Contig Fragment Size (12) the sequential base numbers are used for base matching. The range of Fragment Size is Mutation Surveyor takes a string of 12 letters of the DNA sequence from a GenBank file, and then compares the text call to the sample and reference files. If it matches to the sample, it will move on to the next window of 12 bases, and makes another comparison. This moving window method is used to check the matching sequence. Larger numbers are ideal since they are the most rigorous. Matching Base Number (60) the total number of bases that matched between two sequences. The range of Matching Base Number is If the number of bases that match between two sequences is greater than 60 (default), then the sequences will be in the same contig. 23

25 Chapter 3 General Procedure Matching Base Percentage (30%) the percentage of matching bases in two sequences. The range of Matching Base Percentage is 20 to 99%. The percentage is relative to the shortest sequence. For example, if there are 235 bases matched between two 256 base sequences, the matching base percentage will be 235/356 = 66%. Since 66% is greater than 30%, the two sequences will be in the same contig. Exclude the 1st Base Difference > X bps: (not selected, 200 as starting point) sorts those lanes with a starting base difference greater than 200 bases into two contigs. For instance, the reference covers bases, sample one covers bases, and the second sample covers bases, then the software would sort them into the same contig. However, it would be very difficult to find mutations, because of the fat peaks at the end of the sequences which are compared to the front sharp peaks. If this option is selected, the software sorts these two samples into two contigs. Force into One Contig (selected) designates the samples to be grouped into one contig. Exact File name F/R Match (not selected) determines the rigor with which filenames are matched. Select this option when filenames are identical except for f/r so that files will be paired correctly. When this option is not selected, the software allows for some slight variations in sample filename when sorting them into forward and reverse pairs. Basepatch (not selected) corrects for poor mobility shift within the sample traces. Calculate Lane Quality Within the Region of Interest (not selected) calculate lane quality for region of interest and not entire trace. Trimming Quality Trim (selected) eliminates low quality data at the beginning and end of a sequence. Data at the beginning and end will automatically be trimmed until processable data is obtained. Quality trimming cuts the data based on the data quality in the mutation electropherogram. Vertical blue lines in the mutation traces indicate the limits of trim range (see image below). Vector Trim (not selected) Vector trimming cuts the data with the user-defined beginning and end sequence. The beginning and ending sequences are input in the SEQ File Editor (Chapter 6 File Editors). 5 /3 Trim enables the user to trim the ends of the sample traces by x bases. In some cases the beginning and the end of the traces have poor quality data. It is recommended to trim the ends of these traces to avoid false positive mutation calls. Score Trim (15) allows the user to reject regions for mutation detection where there are 7 consecutive bases out of a group of 9 bases with a score below the defined value. When the score trim is set to 10 for example, and there are 7 consecutive bases with a score below 10 in a group of 9 bases, then the software will disregard this region for mutation detection. 24

26 Chapter 3 General Procedure Contig Sort By Sample/Reference Filename/Contig Num. (Reference Filename) controls the order of contigs within a project, if multiple contigs are present. Sorting may be on the basis of sample filename, reference filename, or the number of sample files in the contig. GenBank Reference Comparison controls the files used for reference in a project. If this option is unchecked, then the software will not use the GenBank sequence as a reference for comparison. Rather, the software will compare the sample traces with the reference files input in the second panel labeled Reference Files. If this option is checked, the software will use the GenBank sequence as a reference for direct comparison to the sample traces. Mutation In the Mutation tab are the parameters by which mutations are called, how their overall score is calculated, and how their confidence rating is determined. Mutation Mutation Score (5.00) is used to call a mutation and rank its confidence level. Mutation score is a measure of the probability of error and is based on the ratios of noise level, overlapping factor and dropping factor. Accuracy is defined as 100% minus the error percentage. In Mutation Surveyor, the highest possible confidence, 99.9% corresponds to a mutation score of 30. A score of 20 corresponds to 99% accuracy; a score of 10 corresponds to 90% accuracy. Mutation Height (500) is the height of the mutation peak above which a mutation is registered. The mutation height threshold is the minimum mutation peak height to be called as a mutation peak. For example, the default setting is 500 for the mutation height. If a peak height in the mutation electropherogram is greater than 500 and other parameters are satisfied, then it is called as a mutation peak. Overlapping Factor (0.20) determines the overlap cutoff point for a mutation to be registered. Overlapping factor is calculated from the two different bases in the reference and sample traces on either side of the mutation. The overlapping factor for an ideal mutation peak is 1.0. In reality, it is normally less than one. For example, if the two bases, AC overlap by 82%, their overlapping factor is Dropping Factor (0.20) determines the dropping factor cutoff point for a mutation to be registered. Dropping factor is the relative intensity drop of the sample trace relative to the reference trace. It is determined from the relative intensities of the four neighboring peaks between sample traces and reference traces; two peaks on each side of the potential mutation. The dropping factor is 1, or 100%, for a homozygous mutation, and it is about 0.5 (50%) for a heterozygous mutation. If there is an artificial peak in the electropherogram due to a bubble in the capillary during electrophoresis, and the artificial peak does not cause any intensity drop of the sample nucleotide, the software will ignore this artifact mutation peak due to its low dropping factor. 25

27 Chapter 3 General Procedure SnRatio (1.00) determines how large the signal has to be relative to neighboring noise in order for a mutation to be registered. Signal is defined as the mutation peak intensity, and noise is defined as the median noise intensity around the mutation peak in the mutation electropherogram. Mutations occur at a rate of one mutation in every 1200 bases in the human genome. If a major peak in the mutation electropherogram is a mutation, the other smaller peaks around this mutation peak in the mutation electropherogram will generally be noise. When the signal (mutation peak) and noise intensities are determined, the probability of the mutation is then based on statistical theory. Some base calling software is able to completely eliminate data noise in the electropherogram, providing extremely clean DNA sequence traces; the software in this software assumes the noise to be 250 RFU. Mobility Shift (imbedded algorithm) is an artifact of sequencing chemistry that can affect mutation identification. All base calling software packages have a function to correct for mobility shift. The different dyes attached to the same DNA fragment will effect the DNA migration differently in gel electrophoresis due to their varying weights. A heterozygous mutation of two colors of the same size molecule will therefore shift in time. Mobility shift is very sensitive to temperature variations over the separation. Unfortunately, sequencing software frequently uses the same set of constants to correct the mobility shift for different capillaries in the same run or for different runs. Therefore mobility shift is not completely corrected due to the temperature variations over the capillaries. The mobility shift often lowers the mutation peak intensity. Therefore, Mutation Surveyor scans for instances when the peak intensity (height) score is greater than 500 (the default cut-off) yet the overall mutation score is low. If the dropping factor reaches 0.15 and another uncalled peak is detected around the major peak, Mutation Surveyor will automatically change the uncalled peak to a called peak in the mutation report. A new mutation score is given after uncalled peak is moved to the center of the called peak. As well, the comment line will display Added by computer. Original data is not changed in this process. Mutation Detection High/Medium Sensitivity (High Sensitivity) determines the level of sensitivity the software uses to call mutations. When high sensitivity is checked, the software can detect mutations in areas of high frequency and noise. Allow Computer to Delete Mutations (selected) allows the software to delete illogical mutations and those with high background noise. Peak Fronting Correction (5%): corrects for chemical artifacts that can lead to missed mutations. Peak fronting is often seen as a smaller peak in front of a major peak of the same color (see image at right). This results from the PCR reactions of a dye-labeled terminator when concentrated reagents are used. This correction factor is used to subtract a minor fronting peak from a major peak when both peaks are in the same color and therefore, the same nucleotide in the sequence. Peak fronting correction may reduce the mutation intensity of some peaks in the mutation electropherogram. 26

28 Chapter 3 General Procedure Base Labeling Peak Center (selected) determines what point on a peak is used for determining peak height. Some base calling programs write the base position at the center of the peak while others label the base position at the edges of the peaks (see image at right). This variation can introduce false positives as samples are aligned with GenBank references or other references generated in a different platform than the samples to which they are being compared. If Peak Center is chosen, nucleotides will be labeled at the peak centers. Using Peak Center can reduce the number of false positives. Original (not selected) the base position will be at the same spot as placed by the original base calling software, such as ABI Basecaller. Data Processing Dye Blob Baseline Removal (not selected) removes baseline noise. In the event the sequence traces exhibits broad peaks caused by a dye blob, the baseline may introduce artifacts into the mutation electropherogram. Baseline removal may also reduce the mutation peak intensity and should be used with discretion. Output The selections made in this section do not affect the analysis parameters in any way; as the title indicates, changes made here affect how data is output. Output Table Fields (defaults are shown selected in figure) specifies those items included in the ORT. Quality Thresholds Lane Quality > = (0) lanes not meeting the lane quality threshold will be placed in a separate folder other than the contig folder. The range of Lane Quality is Output Limits CDS Only (not selected) Graphical output limited to CDS. Before (After) CDS (available when CDS Only is selected) select or limit the number of bases preceding or following the exon. Region of Interest Only (not selected) Graphical output limited to Region of Interest. NOTE: CDS and ROI can be designated in the SEQ and GBK File Editors (Chapter 6 File Editors). Print Default Header Information (selected) resets Header to default settings. Display Four very different aspects of Mutation Surveyor are controlled in this panel. Mutation Call Thresholds The parameters involved have been discussed in detail in Chapter 4 Mutations. 27

29 Chapter 3 General Procedure Mutation Call Thresholds determines if an identified mutation is displayed as red - low confidence - or blue - high confidence. These colors are used as a way to alert users that a mutation peak may not be reliable. The numbers in the left column indicate the lower rejection boundary; if the number is smaller than this number; the mutation will be rejected and not displayed. If the mutation falls into the range of the two numbers, it will be accepted but with the red alert color. If the mutation parameters are greater than the pass settings in the right column they are marked with the high confidence color of blue. The example shown at right indicates 1D settings. For 2D settings, the levels are twice the 1D levels. Amino Acid 1/3 Letters (1 Letter) determines if amino acids are noted with their one or three letter code. Both codes are listed in Chapter 2 Features Overview. With Indel Change (selected) allows the customer to choose amino acid output including or excluding indels Check 2D Small Peaks (Mosaic) Check (not selected) If activated the software will search for peaks buried within the baseline and indicate their presence with a short green bar if they are of the same wavelength and are in the same spatial position in both strands of sequence data. Height Threshold (available when Check is selected - 100) set RFU value for the smallest peak to be detected. Maximum Height in Mutation Electropherogram (5000) if the heights of peaks in the trace are over this set Maximum Height, then the software will normalize the peak to this Maximum Height. Display Phred Score (not selected) when selected, the Phred score for each peak will be displayed in the Sample trace directly below the base call. Display Negative SNP (not selected) when selected, the GAD will display locations with reported variations that do not contain the SNP by showing the possible alleles in green font at the top of the mutation electropherogram. Reports will display the negative SNP in green font. 2 Directions The parameters in the 2 Directions tab determine how Mutation Surveyor will examine mutations. It is critical that the correct setting be used - incorrect configuration can greatly affect mutation identification. Settings 2 Directions should be selected when paired; forward and reverse data has been entered. If two strands of DNA sequences are compared and the two mutation peaks are at the same location in both strands, it is generally a real mutation. If there is a peak in one strand of the (forward or reverse) DNA sequence, then it is generally not a real mutation. Mutation score thresholds for both the forward and reverse sequences are additive. For example if the pass score is set at <20, the forward direction has a score of 2 and the reverse score is 20 then the sum score will 22 and the mutation will be accepted. 1 Direction should be used for data in only one direction. This is the basic, default setting for the entire analysis package. A single analysis is done on one mutation score. 28