DNASIS MAX V2.0. Tutorial Booklet

Transcription

1 Sequence Analysis Software DNASIS MAX V2.0 Tutorial Booklet

2 CONTENTS Introduction DNASIS MAX : Protein Translation & Function : Nucleic Acid Alignments(BLAST Search) : Vector Trimming : Entrez Search : Managing an In-House Databases : Restriction Enzyme Site Search DNASpace : Automated Sequence Grouping : GenBank Report Batch Retrieval Contig Manager : Linking Trace Data Phred/Phrap : Introduction : Linking Trace data : Using Quality Values for Analysis..52 1

3 Introduction The DNASIS MAX 2.0 Tutorial Booklet describes the step-by-step procedures to successfully use each of the software s main features. This booklet is divided into three main sections corresponding to the main sections of the software: 1) DNASIS MAX, 2) DNASpace, and 3) Contig Manager. Please note the following before first using the product. * Based on copyright laws, any portion, or the entirety of this product (including the Reference Manual and software) cannot be copied or transferred. *All brand names and product names described in this manual are trademarks or registered trademarks of their respective companies. Operating Environment DNASIS MAX system requirements are described below. HARDWARE Pentium or higher microprocessor on a DOS/V Computer. P III 600 MHz is recommended for optimal performance. 128 MB RAM or more. 100 MB hard disk memory. To install all sample sequence databases, you will require an additional 400 MB. CD-ROM drive 1024 x 768 display with 256-colors. Internet connection for external database searchers SOFTWARE Windows 98, ME, 2000, XP, or NT4.0 SP6 or higher Internet Explorer 4.01 or higher (Even if you are not online). Usage Restrictions The DNASIS MAX trial version provides all the features of the full-product version except for saving, printing, and copying. Note, the trial version will expire 30 days after installation. Installation If a previous trial version of DNASIS MAX is installed, please uninstall it first. If you are using Windows NT, 2000, or XP, you will need to log in with administrator privileges to install DNASIS MAX. Installer program for DNASIS MAX Trial consists of four files described below: 1. DNASIS_MAX_Trial_V2.1.2.exe 2. Sample_Database_for_DNASIS_MAX.exe 3. MSDE2000_for_DNASIS_MAX_Trial.exe 4. Contig_Manager_Trial_V2.1.2.exe Download these files from MiraiBio s website and double click them sequentially. No. 3 and No.4 are optional for sequence assemble function. After installing MSDE 2000, be sure to install the latest patch from MiraiBio s website Tutorial Data Once the Setup program is launched, Most of the default installation settings will be sufficient. However, selecting a database directory will require you to enter the database name correctly. All tutorial data, sample sequence database, and the genome database will be installed in this directory (Note: this will take up approximately 400 MB of memory). Please note, the directory name cannot contain any spaces. To open the folder with the tutorial data, go to the Windows Start menu and choose Program/DNASIS MAX Trial V2.0/Tutorial data. Data sets for each tutorial are located here. If you chose the default values during 2

4 installation, the data installed in C:\HSK_DB_Trial is as shown in Figure 3. AAMotifDB BlastDB GenomeDB Folder Name Contents Motif database of amino acids. BLAST search database. Entire genome sequence database for E.coli K-12 MG1655. Multiple alignment profile MultipleAlignmentProfile database. NAMotifDB SequenceDB TutorialData VectorData Figure 3: Databases Starting DNASIS MAX Nucleic Acid Motif database. Sequence database. Sample data used in tutorials. Vector data files that can be registered in vector databases. From the Windows Start menu, choose Program / DNASIS MAX Trial V2.0 / DNASIS MAX. The screen shown in Figure 4 will appear. Figure 4: DNASIS MAX Launch Screen After initialization, DNASIS MAX launches, and the initial entry screen appears, as shown in Figure 5. Figure 5: Main DNASIS MAX Screen Data Comment View Map View Sequence View Analysis Button View Screen Toggle Toolbar Internet Connectivity DNASIS MAX relies on Internet connectivity for many database search features. As such, it is highly recommended that it be used with an Internet connection. You will need to set up a few parameters before you first use DNASIS online. First, select View / Internet Options. The screen shown in Figure 6 will appear. Here, you will set up an HTTP proxy server. Figure 6: HTTP Proxy Configuration Screen 3

5 For details on editing HTTP Proxy settings, ask your network administrator. The Internet Parameters window also includes FTP Firewall and Mail settings, which are not supported in this version. Contact Information Contact information for MiraiBio is noted below. MiraiBio, Inc Harbor Bay Parkway Ste.150 Alameda, CA Toll-Free (USA Only): Phone: Fax: Web: 4

6 1. DNASIS MAX 1-1: Protein Translation & Function The Protein Translation & Function section describes how DNASIS MAX converts a genetic sequence to the potential proteins and then determines the possible functionality of each motif within the protein sequence. Figure 1-1: Sequence Editor Screen This process is done by performing the following basic functions: 1) Open Reading Frame (ORF) search, 2) Translation, and 3) Amino Acid Motif Search. Opening the Sequence File This tutorial uses Tutorial1.fsa as the entry sequence. From the main DNASIS MAX window, select the Open option, and choose Tutorial1.fsa. Refer to the Introduction for the location of tutorial sequences and data. You can also use the methods below to open the sequence file. Performing an ORF Search From the Analysis Button view, choose the DNA search group. Figure 1-2: ORF Button ORF Button Click the Open button ( ) from the toolbar and select the sequence file. Choose the sequence file with your mouse, drag it to the DNASIS MAX main screen, and release. Each sequence file format is automatically determined and entered into the editor. File formats that can be opened in the editor are GenBank, EMBL, PIR, Fasta, DNASIS, text, and trace (ABI, SCF). When the ORF button is clicked, an ORF search is performed starting with any of the first three bases. Once the analysis is complete, the results are displayed in the sequence view page, and map view page (Figure 1-3). Each ORF is noted with an arrow. From the Analysis button view, you can change the Codon Table by clicking your right mouse button while hovering above the ORF button, and selecting the parameter menu (Figure 1-4). 5

7 Figure 1-3: ORF Search Results Table as needed. Figure 1-6: Codon Table Editor Figure 1-4: Choosing the Parameter Menu You can also specify the initial codon with the Initial Codons option from the drop-down list. Performing Translations To perform translations, first select the foundation DNA group from the Analysis Button view. Figure 1-7: Translation Button 1-5. The Parameter Settings dialog opens as shown in Figure Figure 1-5: Parameter Settings Dialog After the translation is conducted in three frames, the results are displayed as shown in Figure 1-8 below. The Codon Table is initially set to Universal. To change it, choose a different table from the drop-down list, and click OK. If you click on the ellipsis button (...) at the right of the Codon Table name, the Codon Table Editor will open (Figure 1-6). You can then edit the individual contents of the Codon 6

8 Figure 1-8: Translation Results Screen format will change to one character. Displaying Only the Longest ORF If there are multiple ORFs, the display may be difficult to read. To solve this, click the right mouse button and choose to display the Show Setting menu within the ORF results (Figure 1-11). Figure 1-11: Show Settings Screen To change the amino acids identification format from three characters to one, click the right mouse button and choose Property/Menu from within the Sequence View window (Figure 1-9). Figure 1-9: Selecting the Property Menu Now, click the Show Top option under ORF, as shown in Figure Here, you will enter how many spaces in length from the longest length to display. So, if you want to display the longest ORF only, enter 1. Figure 1-12: ORF List Display Screen The Parameter Settings screen will open (Figure 1-10). Figure 1-10: Parameter Settings Screen To save the ORF list, choose File-Export from the menu. This lets you save the data in the list displayed as a Under Amino Acid Symbols, choose the option for one letter, and click the OK button. The selected amino acid identifier tab-delineated text file. 7

9 Entering ORF Amino Acid Sequences When you click on the longest ORF in the Sequence View, the corresponding amino acid sequence will be highlighted. In this case, the first frame's amino acid sequence will be highlighted (Figure 1-13). Running an Amino Acid Motif Search Choose the Amino Acid search group from the Analysis Button View and click the Motif Search button, shown in Figure Figure 1-15: Amino Acid Motif Search Figure 1-13: ORF Selection Screen The search is conducted against the motifs registered in the Amino Acid Motif Database. Results are displayed in Sequence View and in Map View (Figure 1-16). Figure 1-16: Amino Acid Motif Search Results Screen When the New Amino Acid Button ( ) is clicked, DNASIS MAX switches to the amino acid mode and displays the new protein sequence (Figure 1-14). Figure 1-14: ORF Amino Acid Entry Screen To see detailed information on a particular motif, double-click on a motif name in the amino acid motif analysis display area. Figure 1-17 displays the resulting window of information on the chosen amino acid motif. To toggle between the DNA sequence and Amino Acid sequence modes, click the DNA Mode button ( ) or click the Amino Acid Mode ( ) button from the Screen Toggle Toolbar at the right side of the software. 8

10 Figure 1-17: Amino AcidMotif Data 9

11 1-2: Nucleic Acid Alignments (BLAST Search) The Nucleic Acid Alignments section describes how DNASIS MAX performs a basic nucleic acid sequence alignment and a comparison to NCBI Entraz databases. Figure 2-2: Parameters Setting Screen This process is done by performing the following basic functions: 1) BLAST search, 2) a multiple sequence alignment and 3) custom annotation Opening a Sequence File This tutorial section uses Tutorial2.fsa data file. From the file menu in the main DNASIS MAX window, choose Open and select the appropriate data file. Specifying the BLAST Search Target Database Prior to running a BLAST search, set the parameters and conditions for the search. Move the cursor over the BLAST Search button (Figure 2-1), click the right mouse button and choose the Parameter menu. Use the default value, blastn, in the Program name field. In the Nucleotide Database field, the MAM (i.e., mammalian) database should be checked. Click OK. Running a BLAST Search When the BLAST Search button is clicked, a BLAST search is run against the MAM database. When the analysis is complete, the results will display, as shown in Figure 2-3, below. Figure 2-3: Homology Search Rsults Screen Figure 2-1: BLAST Search Button BLAST Search Button Homologous Sequence Search The BLAST Search Parameters Setting dialog will open as shown in Figure 2-2. From the results list, choose the result with the highest similarity hit, M98484 AALMTCYTOB, from the BLAST search and click the Get GenBank Report button (Figure 2-4). 10

12 Figure 2-4: Get GenBank Report Button The alignment will be conducted using the ClustalW method between two sequences. The results will display within the editor, as in Figure 2-7 below. Figure 2-7: Multiple Alignment Results Screen Get GenBank Report Button The system accesses the NCB Entrez system via the Internet to obtain the access number M98484 in GenBank. When a homolgous sequence is found, DNASIS MAX will disply this as a newly defined sequence (Figure 2-5). Figure 2-5: Sequences Obtained Screen Adding Annotations from the homologous regions of the two sequences are displayed on a yellow background. For this exercise, an annotation will be added to the unknown sequence for the matching region between 301bp to 540bp matches,. First select/highlight the area from 301bp to 540bp by left clicking the mouse over base 301 in the Sequence View and drage to the 540 bp (Figure 2-8). Figure 2-8: Screen With Common Areas Selected Conducting Multiple Alignments In the Analysis Button View, choose the DNA Multiple Sequence group and click the Multiple Alignment button (Figure 2-6). Figure 2-6: Multiple Alignment Button 11

13 Next, click the Alignment Mode button ( ) on the Screen Toggle Toolbar to add annotation to the unknown sequence. The unknown sequence (Tutorial Data 2) must be the only sequence highlighted before annotation. To do this, simply click on the highlighted region (pink/magenta) of the M98984 sequence in the Map View section of the DNASIS MAX main screen. Now, click the New Annotation Entry Figure 2-10: The Added Annotation button ( ). Once the Annotation Setting screen opens (Figure 2-9), enter the annotation name, Homology with M98484AALMTCYTOB in the Annotation name field. Then, click the OK button. Figure 2-9: Annotation Addition Screen In the Sequence View section of the DNASIS MAX main screen, the annotation Homology with M98484 AALMTCYTOB will be added (Figure 2-10). 12

14 1-3: Vector Trimming Figure 3-2: Vector Database Manager Screen The Vector Trimming section describes how DNASIS MAX performs the procedures needed to eliminate a vector s sequence from the target/entry sequence and perform an alignment. This process is done by adding a known vector sequence to a vector database, trimming the target/entry sequence, masking the vector sequence and performing an alignment with a reference sequence. Opening a Sequence File This tutorial section uses Tutorial3_1.abi data file. From the file menu in the main DNASIS MAX window, choose Open and select the appropriate data file. Vector Trimming From the Analysis Button View, select DNA - Basic, right mouse click the Trimming Button (Figure 3-3). And choose the parameter item. Figure 3-3: Vector Trimming Button Registering the Vector Sequence Since trimming is routinely done, it is best to register vector sequences in DNASIS MAX s built-in database. To do this, select the Database Tab in Analysis Button View and click the Vector Database button (Figure 3-1). Figure 3-1: Vector Database Button After clicking on the Parameter menu item, the Sequence Trimming Parameter Editor opens (Figure 3-4). Figure 3-4: Trimming Parameter Settings Vector Database Button The Vector Database Manager screen opens (Figure 3-2). To add a vector sequence to the vector database, click the Import Button, and import the file, psu2718.prm (an example of a vector sequence), from C:\HSK_DB_Trial\VectorData. The vector sequence, psu2718.prm, is now registered in the vector database. In the Trim Vector section of this window, highlight psu2718 from the Vector Name list. Next, choose SmaI from the Cloning List and click the OK button. Click the Trimming button. If the vector sequence is discovered (Figure 3-5) in the target/entry sequence, the Sequence View will show the 13

15 vector sequence area will and label it (in this example, psu2718[sma]). Figure 3-7: Trace View Figure 3-5: Trimming Results Screen Masking the Vector Sequence In the Sequence View, click the Vector (psu2718[smai]), and the vector sequence will be highlighted. To mask the vector sequence, click the Mask button ( ) in the toolbar,. This replaces the vector sequence with multiple N s, which signifies a masked (Figure 3-6). To select a reference sequence for alignment comparison,, first click the Import Alignment Sequence button ( ). For this example, highlight the Tutorial3_2.fsa sequence as a reference and click Import. Upon importing, an automatic ClutsalW alignment is performed showing all differences in blue highlight (Figure 3-8). Figure 3-8: Reference Sequence Screen Figure 3-6: Masking Results Specifying a Reference Sequence To conveniently compare the masked target sequence with a reference sequence, it is beneficial to view in the Trace View. On the Screen Toggle toolbar, click the Trace View button ( ) to view the trace of the target/entry sequence (Figure 3-7). Reference Sequence Alignment Click the Show Alignments button ( ) to conduct an alignment between the target/entry and reference sequences. This alignment also uses a ClutsalW alignment. Any resulting differences, possible mutation locations, are again highlighted in blue. This tutorial demonstrates two such mutations at locations 59bp and 74bp. 14

16 Figure 3-9: Reference Sequence Screen 15

17 1-4: Entrez Search The Entrez Search Section describes how DNASIS MAX performs an NCBI search, via the Internet, and the best way to display the results. Running an Entrez Search From the Analysis Button View, choose Database and click on the NCBI Entrez button (Figure 4-1). Figure 4-1: NCBI Entrez Search Button Table 4-1: Specifying Keywords # Search Target Field Conditional Value 1 All Fields is p53 2 Organism is human Figure 4-3: Keyword Selection Example After clicking the Search button, the system connects to NCBI and runs the search. The results are displayed in the Search Results (Figure 4-4). Figure 4-4: Entrez Search Display Screen The GenBank Keyword Search dialog box opens (Figure 4-2). Figure 4-2: Entrez Search Keyword Selection Screen Adding Results to DNASIS MAX Once the NCBI Entrez search is completed, any of the resulting sequences can be added to the Sequence View of In this dialog box, choose Nucleotides in the Database field. Refer to Table 4-1 below to set the keyword conditions for this search. To add the second keyword search condition, click on the New Keyword button. Also, select AND in the logic field DNASIS MAX for further work. First highlight the result of interest. In this example, click the sequence result with an accession number NM_ (Figure 4-5). Figure 4-5: GenBank Report Button for this example (Figure 4.3). The last step is to click the Search button. To add this to DNASIS MAX Sequence View, click on the GenBank Report button (Figure 4-5). You can also 16

18 double-click the appropriate sequence to enter into DNASIS MAX (Figure 4-6). Finally, you can also use the Shift and Control keyboard keys to select multiple sequences of interest and import them into the Sequence View. Saving Entrez Search Results Entrez Search results are saved in your project file, and you can reference these results even if you close the Entrez search result window. To do this, close the previous Entrez search result window. From the DNASIS MAX right toolbar, Figure 4-6: Sequence Capture Screen click the button. Entrez search results will be stored in the comprehensive data view dialog, shown in Figure Figure 2-4-9: Comprehensive Data View Dialog To view Entrez search results again, choose the Entrez search data, and click the Open button. Figure : Entrez Search Results Retrieval Managing Annotation Data When working with multiple sequences, annotations and/or analysis results can become distracting. To annotation information, right click Annotation for the NM_ sequence. Clicking on the Hide Analysis menu item (Figure 4-7) will hide all annotations for this sequence. The annotations will no longer be displayed. Figure 4-7: Hiding Annotation Data To display annotations again, you can either right-click in the open space between Accession numbers from the Sequence View or click the Data List button in Toggle Tool bar. Highlight the annotation row of interest, click the Show button, and then click OK. The annotations previously hidden for NM_ will reappear. 17

19 1-5: Managing an In-House Databases Figure 2-5-2: New Database dialog In a BLAST search, you can specify public databases, such as GenBank, or an in-house customized database. DNASIS MAX also provides the user with the ability to search, renew, and delete customized databases. Creating an In-House Database To create your own customized database, follow the steps below. 1. Launch DNASIS MAX. Figure 2-5-3: Specifying the Data Source 2. From the database tab in the analysis button view, click Sequence Database to launch the Sequence Database Manager, shown in Figure Click New. The New Database dialog will display, as shown in Figure Click the In-House tab. 5. Under the Name column, enter Tutorial4. 6. Make sure that the data source value is Blank(Nucleotides), as shown in Figure Click the OK button to close the dialog box. 8. Check that in the Sequence Database Manager list, a Tutorial4 database with entry number 0 has been created. 9. Click the OK button to close the dialog. Figure 2-5-1: Sequence Database Manager Registering Sequence Entries You have now created a blank sequence database. Now, you need to register sequence entries. To do so, follow the steps below. 1. Enter the data to be registered in the DNASIS MAX main screen. Here, open the folder containing tutorial data (TutorialData) and open the ContigManager / TutodialData1 / TraceData1_1 folder. Select all 30 items, and drag and drop to the DNASIS MAX main screen, as shown in Figure From the database tab in the analysis button view, right-click on the In-House Database registration (DNA Sequence) button and choose the Parameter option from the pop-up menu. The dialog shown in Figure will appear. 3. Choose the Tutorial4 database. Make sure the options Update related BLAST database after updating and 18

20 Make new BLAST database if it doesn't exist are both checked, as shown in Figure Click the OK button to close the dialog. 5. Now, left-click on the In-House Database registration (DNA Sequence) button while pressing the Control key. All 30 sequences displayed on the screen will be registered as a batch in the Tutorial4 database. The Control key is used to register the sequences as a batch. If you do not use the Control key, only the current analysis target sequence Figure To view registration details, choose Tutorial4, and click the View button. You can now view the entries in detail, as shown in Figure Use the scroll bar to switch from one entry to another. 4. Click the Close button to close the window. 5. Click the OK button to close the dialog. Figure 2-5-6: Successful Registration (the item with an underlying bar) will be registered. Figure 2-5-4: Selecting Items for Registration Figure 2-5-7: Registration Entry Details Figure 2-5-5: Setting Parameters for In-House DB Registration Conducting a BLAST Search Next, conduct a BLAST search using the database created. Follow the steps below. To ensure that the sequences are registered, follow the steps below. 1. From the analysis button view, database tab, click Sequence Database. 2. Check that the 30 items are indeed registered, as shown in 1. From the analysis button view, choose the Compare DNA tab. Right-click the BLAST Search button, and choose the Parameter option from the pop-up menu. The window for setting BLAST parameters, shown in Figure 2-5-8, will appear. 2. Click the Deselect All button to clear settings. 3. From the database list, find the Tutorial4 database 19

21 previously created, and mark it with a check, as shown in Figure Figure : BLAST Search Results Window 4. Click the OK button to close the dialog. 5. In the topmost sequence in the window, click the name, It will be displayed with an underlying bar, indicating that it is the current analysis target sequence, as shown in Figure Now, left-click the BLAST search button to run a BLAST search. Search results will display in a separate window, shown in Figure Figure 2-5-8: Specifying a Target Database Note that in an In-house DB BLAST search, the query sequence itself is removed from the BLAST search results, and displayed separately. Searching, Renewing, and Removing an In-house Database The procedures for maintaining your customized in-house Figure 2-5-9: Displaying the Target Sequence database are outlined below. 1. From the analysis button view, choose the database tab, and click the Sequence Database option. 2. Choose Tutorial4, and click the View button. 3. Choose Entry / Find from the menu. The Search dialog, Figure , will display. 4. Choose Definition from the Find in drop-down list, and enter the value for the Find What field. 5. Click the Find button. The corresponding entries will be searched and displayed automatically, as shown in Figure Figure : Search Dialog 20

22 Figure : Search Results Display window. The top hit entry definition should be the new value entered previously, TEST. Figure : BLAST Database Manager To change other search parameters, follow the steps below. 1. Choose the Entry / Property menu to launch the search dialog, shown in Figure You can edit the data source and definitions here. 2. Try setting the definition value to TEST. 3. Click OK to close the dialog. In this state, the BLAST database has not been modified. The BLAST database is a modified version of the Sequence database, and is designed for running rapid BLAST searches. A BLAST database is always required. For sequence registration, the BLAST database is updated automatically, but for renewing and deleting sequences, you will need to manually update the BLAST database. To do so, follow the steps below. 1. From the Analysis button view, choose the database tab, and click on Dedicated BLAST Search Database. The BLAST Database Manager launches, as shown in Figure Choose the Tutorial4 database, and click the Update button. 3. Click OK to close the dialog. 4. Try conducting another BLAST search by clicking the BLAST search button. Results will display in a separate 21

23 1-6: Restriction Enzyme Site Search This tutorial discusses the restriction enzyme site search feature. Topics covered include the following: methodology for choosing a restriction enzyme, viewing the results display, the cut location view screen, the fragment list display screen, cut location list by restriction enzyme, choosing the optimal restriction enzyme for cutting at a specific interval, obtaining a list of restriction enzymes that do not cut the sequence, and using multiple restriction enzyme sets for searches. Running a Restriction Enzyme Site Search Click the restriction enzyme site search button. The results will display in a window, as shown in Figure In the map view, cut locations are indicated with a pin. Figure 2-6-2: Restriction Enzyme Site Search Results Display Window Opening a Sequence File This tutorial uses Tutorial1.fsa as the entry sequence. From the File menu in the Sequence editor, click Open, and choose Tutorial1.fsa. Choosing an Restriction Enzyme To choose the restriction enzyme for analysis, follow the steps below. 1. First, from the analysis button view DNA search tab, right-click the restriction enzyme site search button. Choose the Parameter option from the pop-up menu. The dialog shown in Figure will appear. 2. Choose the option, Select from a Category. 3. Select the option Recognition Length, and check 4base-cutter. Un-check the Cut Kind option. 4. Click the OK button to close the dialog. Figure 2-6-1: Dialog for Setting Restriction Enzyme In the sequence view at the bottom part of the window, detailed cut locations are indicated atop the sequence. Click the restriction enzyme label to turn the label pink and highlight it, and also to show the cut types with a red line. All cut locations from the same restriction enzyme will all be selected, and displayed in orange. Cut Location List From the restriction enzyme search results graphic view, right-click and choose the Restriction Site List menu from the pop-up menu. The dialog shown in Figure will appear. Figure 2-6-3: Cut Locations Search Parameters 22

24 Moving from the left of the window, the restriction enzyme name, recognition pattern, recognition sequence length, cut type, cut site location, and the cut location are displayed. You can also export the file, or copy the contents to a clipboard. To do this, either click the button circled in Figure 2-6-3, or use the pull-down menu option. Also, you can uncheck the left marks to hide the corresponding cut locations for the restriction enzyme. Click the OK button to close the dialog. Fragment List From the restriction enzyme search results graphic view, right-click and choose the Fragment List menu from the pop-up menu. The dialog shown in Figure will appear. Figure 2-6-4: Fragment List as shown in Figure Choosing the Optimum Restriction Enzyme for a Particular Range It is possible to choose one or a pair of restriction enzymes that includes a particular range and yet does not cut within that range. To do this, follow the steps outlined below. 1. Choose Edit / Select Range menu, and enter 590bp ~790bp. You can also specify a different range. 2. From the restriction enzyme search results view, right click and choose the Search Optimum Enzyme from the pop-up menu. The dialog shown in Figure will appear. 3. Choose the option, 1 or 2 enzymes. 4. Click the Search button. The results will display in a window as shown in Figure The restriction enzymes Tru1I and Hin61 are optimal, and are indicated in red. You can check the map view to make sure that the selected range is included, yet is not cut, and the restriction enzyme closest to the selected range has been chosen. Figure 2-6-5: Cut Location List by Restriction Enzyme You can export the file, or copy the contents to a clipboard. Click the Cancel button to close the dialog. Cut Location List By Restriction Enzyme The map view in the upper window displays all cut locations for all restriction enzymes. You can choose to display by restriction enzyme. To do this, from the restriction enzyme search results view, right-click and choose Separate from the pop-up menu. The display will be per restriction enzyme, as shown in Figure The black lines above each restriction enzyme bar indicate the cut location. Click above the bar to select the cut fragment for each restriction enzyme. The fragment display will change to pink and be highlighted, 23

25 Figure 2-6-6: Highlighting Cut Fragments 790bp. You can also specify a different range. 2. From the analysis button view, choose the DNA Search tab, and right-click the Restriction Enzyme Search button. Choose the Parameter option from the pop-up menu. The dialog shown in Figure will appear. 3. Choose the option, Select from a List. 4. Click the button to the right of User Selected. The dialog shown in Figure will appear. Click the Check All button. 5. Click the OK button to close the dialog. 6. Set the number of cutting sites per enzyme to Min=0 and Figure 2-6-7: Dialog for Choosing the Optimum Restriction Enzyme Max=0. Mark the check box next to Max, as shown in Figure Click the Restriction Enzyme Search button to start analysis. When complete, the system indicates duplicate results, as in Figure Click Overwrite at this point. 8. A blank result without any cut sites will overwrite the Figure 2-6-8: Optimum Restriction Enzyme Search Results previous analysis and display. 9. In the sequence view, right-click on the area with the Enzyme not found message. A pop-up menu will appear. Choose Restriction Site List from the options. 10. A new list, including restriction enzymes that do not have any cut sites (that is, those that do not fragment the sequence at all) will be displayed, as shown in Figure Figure 2-6-9: The Restriction Enzyme List Manager Obtaining a List of Restriction Enzymes that Do Not Fragment a Sequence It is possible to search for restriction enzymes that do not fragment a sequence at all. To do this, follow the steps outlined below. 1. Choose the option for a particular range. From the Edit menu, choose the Select Range menu, and choose 590bp~ 24

26 Figure : Specifying the Number of Cutting Sites per Enzyme Using Multiple Restriction Enzyme Sets You can specify restriction enzyme sets that are frequently used. To do this, use the duplication feature in the analysis button to create new buttons. This section covers creating a button that uses the 5base-cutter category in a restriction enzyme set for analysis. First, right-click above the DNA-search Restriction Enzyme Site Search button. A pop-up menu will appear. Choose the Duplicate option, as shown in Figure Figure : Duplicating the Analysis Button Figure : Overwriting Previous Search Results Figure : Restriction Enzymes with No Cuts List As shown in Figure , the restriction enzyme site search button has been copied. Now, right-click above the button, and choose the Rename option, and rename it as Restriction Enzyme Site Search 5 base cutter. Figure : Screen After Button Duplication Figure : Final List of Restriction Enzymes that Do Not Fragment a Sequence Now, set the button parameters to be in the 5 base cutter category. Try running a search using the Restriction Enzyme Site Search, 5Base-Cutter button. As shown in Figure , note that different parameters have been used for the restriction enzyme site search. 25

27 Figure : Results after Using Different Parameters 26

28 2. DNASpace Figure 3-4: DNASpace Main Screen Launching DNASpace To launch DNASpace, first launch DNASIS MAX. From the Analysis button view, click the DNASpace options group button. You can also launch DNASpace from the Windows start menu, by choosing Program / DNASIS MAX Trial V2.0 / DNASpace Trial. Figure 3-1: DNASpace Button Tree View Space Window List View Main Window Application Palette The icons along the application palette launch various applications, such as homology search and ORF search. Try moving your mouse cursor over the application icons. The The screen shown in Figure 3-2 below will display during initialization. Figure 3-2: DNASpace Launch application name will be displayed in the status bar located at the lower part of the main window. Also, note that various applications are organized by tabs in the lower section of the palette. By dragging and dropping application components to the space window tree view, you will create spaces, or collections of procedures. Creating Spaces Let's try creating a space with these various applications. This example will create a space that runs an ORF search. Once initialization is complete, DNASpace will launch, and the space selection screen shown in Figure 3-3 will appear. Here, click the Close button to close the screen. Figure 3-3: DNASpace Selection Screen First, hold down your left mouse button on the Folder icon at the left side of the application palette and move the mouse cursor to the desired location in the tree view (this process is called drag-and-drop). A new folder will be created in the tree view pane, as shown in Figure 3-5. Figure 3-5: Creating Spaces Names of Various Components The main screen for DNASpace is shown in Figure

29 Next, double-click the newly created Folder icon. The screen shown in Figure 3-6 will appear. Change the name of the folder to Folder1. Figure 3-6: Folder Set Up Screen space. A Parameter Settings screen will appear, as in Figure 3-8. Figure 3-8: Parameter Settings Screen Click the OK button to close the window. Now, drag the Convert Sequence To Fasta application by dragging its icon (second from the left), and drop it in the Folder1 icon in the tree view. Under Folder1, the icon Convert Sequence To Fasta will be linked. Note that when dragging and dropping, the mouse button must be released atop the icon or its name. Link applications in this manner until a space as outlined in Figure 3-7 has been created. The application components are listed in Graph 3-1. Figure 3-7: ORF Search Space The parameters contained within the Parameters frame alter the application's functionality. Here, change the SequenceMode to Both. Now, the ORF search will run on complementary strands also. Click OK to close the Parameter Settings Screen. Now, right-click the Open Reading Frame icon, and choose the System Property option from the pop-up menu. A Settings screen, as shown in Figure 3-9, will appear. Set the AutoOpenView option to true. This sets ORF results to display automatically. (The initial default is to not show ORF results.) Click the OK button to close the System Property Settings Dialog. Now, the space has been created. Graph 3-1: Application Components Figure 3-9: System Property Settings Screen Icon Application Tab Location Convert Sequence To Fasta General (2) Open Reading Frame DNA (2) Open Reading Frame Viewer DNA (3) In the graph above, the numbers after the tab location indicates the number of places the application is from the left. Setting Parameters Now, double-click the Open Reading Frame icon above the 28

30 Be sure to provide a descriptive name for your space, to distinguish from predefined spaces, which are named Space 1, Space 2, and so on. From the Space window menu, choose Space / Rename. Enter a descriptive name (you can enter Japanese names, if desired), and click the OK button to confirm. Running a Space Now, let's try running the space previously created wit the test data available. The test data is included in the initial TutorialData directory specified during system installation. If you choose the default values, the file is at C:\HSK_DB_Trial\TutorialData. From this, choose the U33203.gb file, and drag-and-drop it to the newly created Folder1 space, as shown in Figure Figure 3-10: Running a Space within the range between the start and end codon (that is, ORF), it will be highlighted in yellow, and the sequence and quality data will be displayed in the upper right side of the window. For the purposes of this tutorial, close the ORF results display screen. How Spaces Work Once you drag-and-drop entry data using Windows Explorer, the data is stored in Folder1, and then sent to the application, Convert Sequence to Fasta. This application takes simple text and GenBank format sequence files, and ABI and ALF sequence trace data, and transforms these files to fasta format. The transformed data is stored in Folder2, with the same name as the entry data. Then, this data is sent to the Open Reading Frame application. This application takes DNA sequence in fasta format and runs an ORF search, exporting the results as a file. This file is stored in Folder3. Finally, the data is sent to the Open Reading Frame Viewer application for graphic output and display, as shown in Figure Managing Data Folder1 includes entry data, Folder2 contains intermediary data in fasta format, and Folder3 includes ORF search output. Try clicking Folder1. The left hand list view will change, as The mouse cursor will transform to an hourglass, and the operation will begin. In a few seconds, the ORF result screen, as shown in Figure 3-11, will display. Figure 3-11: ORF Results Display Screen shown in Figure 3-13, and a list of all the contents in Folder1 will display. Double-click the dataset U The data will be displayed in GenBank format. Next, click Folder2. It should contain one item, U Note that this is a different dataset from that in Folder1, because it has been converted to the fasta format. Next, click Folder3. It should contain the same U If you double-click the dataset, an ORF search result screen will display, as shown in Figure Also, right-click on the dataset, and choose the Raw Data option from the menu. A The reverse red triangles indicate the start codon, and the green vertical lines indicate the ending codon. If you click screen, as shown in Figure 3-12, will appear. This is the raw data produced by Open reading Frame. The 29

31 graphic display from double-clicking on the dataset, as shown in Figure 3-11, can be considered a more understandable version of this raw data. As you can see, because the processed data is saved immediately under the application that processed it, viewing results in reverse order of processing is quite easy. Application Types DNASpace supports three basic types of applications. The first is the folder, the repository for datasets. The second is a method, and includes searches and data translations. In Figure 3-7, Convert to fasta and Open reading Frame are of this type. The third type is the View, for representing data graphically. In Figure 3-7, Open Reading Frame Viewer is of this type. DNASIS MAX applications all fall into one of these categories. Folders use the folder icon, and the View icon uses the window as a motif. Figure 3-12: Raw Data Display Figure 3-13: Folder Contents Display As shown above, DNASpace uses the concept of a space to encapsulate procedural steps to process data, saves the results in folders, and uses View modes to display the results. 30

32 DNASpace includes a few pre-constructed spaces, some discussed in detail below. You can also try constructing similar spaces on your own. Refer to the previous section for instructions on creating your own spaces. 2-1: Automated Sequence Grouping This space uses trace data output from the sequencer, runs a trimming, a quality check, and homology search, and groups results into unusable, unknown, and already known. Known*** folder. Using the homology search results, these sequences have been determined as already known by selection by homology results. Viewing Results For example the sequence AF has been determined as already known. To see what homology properties it contains, you can check the homology search results by clicking the ***Search Results*** folder, and displaying the contents, and double-clicking on the AF dataset. The Opening the Space From the main window menu, choose File / Open Space to display the space selection screen, shown in Figure 3-3. Here, Homology Search Results display, as shown in Figure will appear. Figure 4-1-2: Homology Search Results Screen choose the option 1-03, Large Sequence Categorizations, and click the Open button. The space shown in Figure will display. Figure 4-1-1: Automated Sequence Categorization Space You can view the entry sequence by double-clicking the Fasta Format Sequence folder, as in Figure As such, entry, intermediary, and output data are easily accessible and viewable. Figure 4-1-3: Sequence Display Screen Running the Space To run this space, choose all of the tutorial data, and drag-and-drop it to the upper left Entry Data folder. The procedure will take about twenty seconds. Then, click the ***Junk2*** folder, which contains categorized data. These sequences have been determined to be an unusable by the Sequence Quality Check application. Now, click the ****Unknown**** folder. After a homology search using BLAST, these sequences have been determined as unknown by homology results selection. Now, click the ***Already Obtaining a GenBank Report From the Homology search results screen, it is possible to obtain a GenBank report with selected alignments. To do this, click the ***Search Results*** folder, and display the data 31

33 contents as a list. Double-click AF087301, and a report will display, as shown in Figure Starting from the bottom of the list display, select the entries you want to obtain reports for, and click the button from the toolbar. The mouse cursor will turn to the hourglass, and the procedure begins. Because the report requires data retrieval from the NCBI site, this may take a while. In a few minutes, the report, as shown in Figure will display. Figure 4-1-4: GenBank Report Display 32

34 2-2: GenBank Report Batch Retrieval This example outlines entering a list of access numbers, and Figure 4-2-2: Retrieval List View obtaining entries with these numbers, from the NCBI Web site. To use this space, you will need an Internet connection. Opening the Space From the main window menu, choose File / Open, for the Space Selection screen. Choose the GenBank Report Batch Retrieve space, and click the Open button. The space shown in Figure will appear. Figure 4-2-1: GenBank Report Batch Retrieval Spaces Scheduling Retrievals Double-click the ***Drop Accession List*** folder to open the folder settings dialog. Check the Stop Folder option, and click the OK button to close the dialog. Drag-and-drop the accession list to the ***Drop Accession List*** folder. At this point, the list is merely stored in this folder without running a search. Next, choose the ***Drop Accession List*** folder and choose the Node / Schedule menu. A Schedule Settings dialog Next, prepare the list of entries to retrieve. In this case, we will use the sample data included in the space. The sample data is included in the left-most folder of the space, and is called Sample List'. Double-click the list. The screen shown will appear, as shown in Figure Select the Dates option for the Method and enter a date and time. Make sure the dates box is checked. Click the OK button to exit the dialog. Figure 4-2-3: Schedule Setting in Figure will appear. This is the list of entries to retrieve. Use this list format style as a template for creating your own entry lists. The data must be simple text. Running the Space Now, let's run this space. Drag-and-drop the sample list to the ***Drop Accession List*** folder. The process will take about thirty seconds. When complete, click the ***GenBank Report*** folder to see the retrieved entries. Retrieval times will be longer if your list is longer, so if you need to obtain a large number of entries, schedule your retrieval at a convenient time, such as night. 33

35 3. Contig Manager Choose sample.bak and hit the Open button. The Project Name dialog will appear, as shown in Figure 5-4. Click the Initialization Using Sample Data To use the sample data, first click the Contig Manager option from the DNASIS MAX analysis button view. OK button. Figure 5-4 Project Name Dialog Figure 5-1: Contig Manager Button In the Open Project dialog, Select Projects column, the name sample will be displayed. This project is the sample file. Choose this file, and click the Open button. Figure 5-5: Sample File Contig Manager will launch, and the Open Project dialog will appear, as shown in Figure 5-2. Figure 5-2 Open Project Dialog The main window will open, and the sample project will display, as shown in Figure 5-6. Figure 5-6: Sample Project Click the Import button. The Open File dialog will display. Here, specify the sample data. If you choose default values when installing, the sample data will be at this location: C:\HSK_DB_Trial\TutorialData\ContigManager\SamplePr oject\sample.bak. Figure 5-3: Sample Data Relinking Sample Trace Data If your installation locations differ from the default, the fragment sequence on-screen will be shown with a question mark icon. This is because the Contig Manager does not retain the trace data within the project, but retains paths to 34

36 externally saved trace data instead. So, if you did not follow the default install, these paths will not work. Figure 5-10: Selecting Files Figure 5-7: Fragments with Broken Links In such cases, Contig Manger must re-link to the actual location of the trace data location. The process is outlined below. From the menu, choose Contig / Relink Trace Files. The Relink Trace Files dialog will display. Figure 5-8: Relink Trace Files Menu In the field Path to a new directory, enter the correct link path. To browse to a folder, click the ellipses button (...), and choose the folder to link to. The initial default location is C:\HSK_DB_Trial\TutorialData\ContigManager\Tutorial \TraceData1-1. Once you specify the folder, the path will change to the folder entered above. Figure 5-11: Choosing a Folder The dialog will show all trace data with broken links. Figure 5-9: Relink Trace File Dialog Once you have selected the folder, click the OK button. The new path will be displayed in the Relink Trace Files dialog. Make sure the path is accurate, and click the Set button. Once the path is correctly identified, all files listed will Choose the data to re-link. To choose all files, choose the disappear, as in Figure topmost file, and while holding down the Shift key, click the bottommost file. 35

37 Figure 5-12: Relink Trace Files Dialog After Setting the Correct Path Make sure no files are displayed, and click the OK button. Figure 5-13: Main Window Now, on the main screen, the fragment sequence icon will be a. 36

38 3-1: Linking Trace Data The tutorial outlines using Contig Manager to retrieve trace data output from the sequencer, and conduct vector trimming and sequence linking. Launching Contig Manager Then, click ContigManager / TutorialData1 / TraceData1_1. Choose all files within the TraceData1-1 folder, and drag-and-drop to either the Root folder in the Contig Manager tree view, or in the list view. If you drag-and-drop in the tree view, be sure to drop it atop the folder to enter data (the folder named Root, in the case of this tutorial). Figure 6-1-3: Entering Sequence Data From the Start menu, choose Program / DNASIS MAX Trial V2.0 / Contig Manager. The Open Project dialog, shown in Figure 6-1-1, will appear. Figure 6-1-1: Open Project Dialog Drag & Drop Once the file has been read, the entire number of data cases, and the read, skipped, and error data names, along Creating a New Project From the dialog, mark next to the Create a New Project button. Enter Tutorial1 for the project name, and click the with the incidence numbers for each, will be displayed in the import summary dialog, shown in Figure Figure 6-1-4: Import Summary Dialog New button. The Contig Manger main window, shown in Figure 6-1-2, will display. Figure Contig Manager Main Window Once you have checked over the fragment contents, click the OK button to close the dialog. Trace Data Display In the Contig Manager main window, a list view will display all fragments entered in the process above. Entering Trace Data From the Start menu, choose Program / DNASIS MAX Trial V2.0 / Tutorial Data to open the TutorialData folder. 37

39 Figure 6-1-5: Fragments in List View Figure 6-1-8: Display of Multiple Trace Data You can double-click a trace fragment to display the corresponding trace data. Figure 6-1-6: Trace Viewer Display of Trace Data Setting Parameters for Trimming 1 Before linking fragments, you can first eliminate vector sequences and sequencing primer sequences with this system. Removing such sequences before linking will allow From the Trace Viewer menu, choose View / Show Single Data, or choose the Single Data button from the toolbar to display trace data in multiple levels, for viewing a wide range of trace data. Figure 6-1-7: Multilevel Trace Data Display for more efficient and productive sequence linking. From the Navigation toolbar choose the Trimming group, and click the Detail button. The Trimming Parameter dialog will appear. Figure 6-1-9: Trimming Parameter Dialog From the Contig Manager list view, choose the option Multiple Fragments, and click the Open button from the Navigation toolbar. This way, you can view multiple trace data simultaneously. This tutorial uses this condition: Trim the first 10bp, while the quality is less than 90%. Also, note that this example does not conduct trimming on registered vector sequences. 38

40 Adding Vector Data Click the Vector Database Manager button from the trimming parameter dialog. The Vector Database Manager, shown in Figure , will launch. This manages registered vector data. Figure : Vector Database Manager data, add the vector to the VectorData folder. The default location is: C:\HSK_DB_Trial\VectorData. If you cannot find this folder, from the start menu, choose Program / DNASIS MAX V2.0 / Tutorial Data, and the topmost layer folder of the folder that opens is where the database is installed. This folder includes more than 900 types of vector data. For details on each vector contents, refer to the file VectorTable.txt in the VectorData folder. Using a spreadsheet program such as MS Excel will help view the contents. Setting Parameters for Trimming 2 Now, let s set the parameters to conduct trimming based on the vector data added in the previous section. Click the Import button. This opens the dialog for importing vectors. Figure : Import Dialog In the trimming parameter dialog, check the option, Trim Vector. Check the box next to the TutorialVectorA option in the Select Vector list, and check the box next to the PrimerA option in the Cloning Sites list. Figure : Vector and Cloning Sites Options Now, find the TutorialVectorA.pm file in the TutorialData folder. If you choose the default options, the location is: C:\HSK_DB_Trial\TutorialData\ContigManager\Tutorial Data1\TutorialVector\TutorialVectorA.prm. Click the Open button. The Vector Database Manager screen will return, with TutorialVetorA at the top of the list. Repeat the process for TutorialVectorB.prm and Similarly, check the boxes next to TutorialVectorB, PrimerB, TutorialVectorC, and PrimerC. If you are actually running an analysis, check the marks next to the cloning site corresponding to the imported vector. This tutorial data establishes the minimum matching percentage considered as contamination at 85%, a bit stricter than the default. TutorialVectorC.prm to add a total of three new vectors. If you are registering a vector that has actually analyzed Click the OK button when the setting is complete. Return 39

41 to the main window, and check the box next to the Trimming option, in the navigation toolbar. click the desired contig from the tree view in the main window. All fragments that comprise the contig selected in the list view and details such as linking directions will display. Now, click Contig1 from the tree view. Contig Viewer will Running an Auto-Assemble To trim and assemble the trace data, follow these steps. In the Contig Manager main window, select all of the launch, and the linking conditions for Contig1 fragments will display graphically, as shown in Figure below. Figure : Graphical Display of Linking Conditions fragments in the list view. (To do this easily, click the desired data from the list view, and simultaneously press down the Control and A keys, or choose Edit / Select All from the Map View menu.) One all the fragments are highlighted, push the Auto Assemble button in the Navigation toolbar. This causes linking to occur, using the base call data contained in the trace data, without consideration of QV values. When complete, the contig sequence data will be added on to the tree view. Figure : Contigs Created Sequence View The Contig screen is divided into two parts. The upper section, map view, includes the entire outline, and the lower section, the sequence view, includes the contig sequence along with each fragment sequence. Comparative Display of Trace Data To evaluate assembled results, it is possible to compare Each fragment's trimming result, and the linking direction details, will be added to the tree view. Figure : New Columns Added to List View actual trace data with various display options. Atop the contig sequence, click the base that you want to display the trace data against. When the base is highlighted, choose View / Chromatograms from the menu, or click the Show Chromatograms button ( ). In Figure , fragment is 951bp in length, Figure : Selecting the Base with trimming at 24bp. In addition, the vector name for trimming is TrutorialVectorA, and once the trimming is complete, is linked by complementary strands. Contig Results Display To display the fragment information of created contig, Using the selected base as the center, the trace data will display together, as shown in Figure

42 Figure : Aligned Trace Data Display Reassembling Once you add a new sequence and include additional fragments, you will need to reassemble. From the Start menu, choose DNASIS MAX Trial V2.0 / Tutorial Data and open the folder. Open the file ContigManager / TutorialData1 / TraceData1_2. Drag and drop the data in this folder to the Root folder. Check the input summary dialog for the correct sequence name and number, and click OK to close the dialog. The trace data display condition will change along corresponding to the selected sequence location atop the Contig Viewer tree view. Click atop the contig sequence in the Contig Viewer, or click the base atop the fragment sequence, to display with the clicked base as the center for displaying multiple trace data together. Note that now, when you choose the Root folder from the tree view, the appropriate fragment is added to the list view. Choose the fragment, and click the Trim button on the Navigation toolbar to conduct vector trimming. If you choose all the fragments in the list view, and click the Assemble button, the confirmation message shown in Figure below will appear. Click Yes, and continue the Figure : Corresponding Display when Clicking a Base from a Contig operation. Figure : Confirmation Dialog The linking procedure will run, including the added fragment, and a new contig sequence will be constructed. Closing Contig Manager Figure : Corresponding Display when Clicking a Base from a Fragment To exit Contig Manager, click File / Exit from the Contig Manager menu. 41

43 42

44 Phred/Phrap Figure 7-1: Phred/Phrap Option Installer Phred/Phrap is an optional feature in DNASIS MAX. Phred/Phrap is an assemble program used worldwide by research facilities and universities engaged in large scale sequencing, and is the de facto standard in the genome analysis field. The feature is designed to read output data from DNA auto sequencers; conduct base calling, and chromatogram quality analysis; and create and edit a consensus sequence. Please note that the DNASIS MAX Phred/Phrap option works with Windows 2000 or Windows XP only. Part 1 Installation and Initial Use Usage Restrictions This trial version of Phred/Phrap supports all functions of the shipping version, but is time limited. The product will be disabled three weeks after installation. Installation To install the Phred/Phrap option Trial, DNASIS MAX should be installed, and launched at least once. Login with Administrator privileges to perform the Phred/Phrap installation. Once you download PhredPhrap_for_DNASIS_MAX_Trial3.exe and double Initialization and Key Code Setup To use the Phred/Phrap feature, you will need a product key. Follow the steps below to start using the feature. 1. Obtain a trial key code by following either of steps. Send an to dnasiskey@miraibio.com, with the subject Phred/Phrap Trial V2.0 Key Code Request. Include these details: your company or organization, department, name, address, phone number, and machine ID (instructions for obtaining this are described below). 2. Enter the product key. A. From the start menu, choose Program / DNASIS MAX Trial V2.0 / Key Code Manager. B. The screen shown in Figure 7-3 will appear. Enter the product key you were sent, and hit the Try button. Figure 7-3: Product Key Entry Screen click it, the installer program shown in Figure 7-1 will launch. Please note that the product key is case sensitive. If the key is entered correctly, the message Success to register key 43

45 code will display; when wrong, the message Invalid key code will display. In this case, check the machine ID and the Project\sample.bak Figure 7-6: Sample Data product key and enter it again. If you still get an error, please contact MiraiBio. Using Sample Data The Phred/Phrap option cannot be used without a valid product key. However, you can still use the sample data to run searches without a product key. To use the sample data, from the DNASIS MAX Analysis button view options, click on Contig Manager and launch Phred/Phrap. Figure 7-4: Contig Manager Button Choose the file sample.bak, and click the Open button. The Project Name dialog will display. Click OK here. Figure 7-7: Project Name Dialog In the Select Projects column in the Open Project dialog box, the project name Sample will display. This project is the sample file. Choose this file, and click the Open button. Contig Manager will launch, and the Open Project dialog will display. Figure 7-5: Open Project Dialog Figure 7-8: Sample File Click on Import. The Open File dialog will display. Choose the sample data here. If you chose the default options during The main window will open, and the sample project will appear. installation, the sample data will be at the location below: C:\HSK_DB_Trial\TutorialData\ContigManager\Sample 44

46 Figure 7-9: Sample Project This dialog contains contig data with broken links. Figure 7-12: Relink Trace File Dialolg Relinking Sample Trace Data If you installed the trial version in locations other than the default, the fragment sequence on screen will be a question mark icon ( ). Because Contig Manager maintains contig Choose the data to Relink. To choose all files, while selecting the top file, hold down the Shift key and click on the bottom file. data not within the project but as links to external contig files, if installed in locations other than the default, these passes are broken. Figure 7-10: Fragments with Broken Links Figure 7-13: Choosing Files In this case, you will need to set up Contig Manager to acknowledge the new contig file locations, in a process known as Relinking. From the menu, choose Contig / Relink Trace Files. The Relink Trace Files dialog will display. Figure 7-11: Relink Trace Files Dialog Enter the correct link path in the box labeled Path to a new directory. To browse to a folder, click the... button, and choose the folder location to link to. The initial folder location is: C:\HSK_DB_Trial\TutorialData\ContigManager\Tutoria ldata1\tracedata1_1 Once you specify the folder, the path will convert to the path above. 45

47 Figure 7-14: Choosing a Folder Make sure no files are displayed, and then click the OK button. Figure 7-16: Main Window The fragment sequences displayed will show a new icon,. Once you have chosen the file, click the OK button. The Relink Trace Files dialog will display that file. Check the path, and click the Set button. If the path has been changed correctly, the fragments listed will disappear. Figure 7-15: Relink Trace Files Dialog 46

48 Part 2 Tutorial Linking Trace Data This section explains using Contig Manager to obtain trace data output from sequencers; conducting base calls and vector trimming using Phred; and sequence linking using Phrap. Launching Contig Manager From the Start menu, choose Program / DNASIS MAX Trial V2.0 / Contig Manager. The Open Project Dialog will Entering Trace Data From the Start menu, choose Program / DNASIS MAX Trial V2.0 / Tutorial Data to open the TutorialData folder. Then, open the ContigManager - TutorialData1 - TraceData1_1 folder. Choose all files in this folder, and drag and drop it to the Contig Manager's tree view Root folder, or to the list view. To drag and drop to the tree view, be sure to select the folder for data to be entered (Root, in this tutorial). Figure 8-1-3: Entering Sequence Data appear. Figure 8-1-1: Open Project Dialog Drag & Drop After the files are transferred, the total number of data Creating a Project In the Open Project dialog, turn the Create a new project button to ON. Enter Tutorial1 for the project name, and sets, the types of data and instances, and types of errors and instances will display in the Import Summary dialog. Figure 8-1-4: Import Summary Dialog click the New button. The Contig Manager main window will open. Figure 8-1-2: Contig Manager Main Window Confirm the entered fragments, and click the OK button to close the dialog. Displaying Trace Data The Contig Manager main window list view shows all the fragments entered above. 47

49 Figure 8-1-5: Fragments in List View Figure 8-1-8: Multiple Trace Data Display Double-click a fragment from the list to view trace data for the corresponding fragment. Figure 8-1-6: Trace Data Display using Trace Viewer Setting Trimming Parameters 1 To make higher quality sequence linkings possible, prior to linking fragments, remove vector sequences and sequencing primer sequences. To do this, Trimming group in the navigation toolbar, click To view a wider range of trace data across multiple levels, click the View Show Single Data option from the Trace Viewer menu, or click the Single Data button the Detail button to display the Trimming Parameters dialog. Figure 8-1-9: Trimming Parameters Dialog ( ) from the toolbar. Figure 8-1-7: Single Trace Data Displayed Across Multiple Levels You can set various parameters for trimming in this dialog. This tutorial is restricted to a unique five or three length base pair sequence, as well as to the condition: Trim the first 10bp, From the Contig Manager list view, choose multiple fragments and then click Open from the toolbar to view multiple trace data in parallel. while the quality is less than 90 %. This tutorial's sample fragment does not conduct trimming on already registered vector sequences, so see the section below, Adding Vector Data, for more information. 48

50 Adding Vector Data In the Trimming Parameters dialog, click the Vector Database Manager button. A management tool for already registered vector data, the Vector Database Manager, will launch. Figure : Vector Database Manager vector from the VectorData folder set up under the Database directory upon installation (the default path is C:\HSK_DB_Trial\VectorData). (If the path is unknown, from the Start menu, choose Program / DNASIS Max Trial V2.0 / Tutorial Data. The folder one level above this is the folder with the database installed.) This folder includes more than 900 types of vector data. To view the contents of each vector, refer to the file VectorTable.txt within each VectorData file. You can use a program such as Excel to view the contents more easily. Once the registration is complete, click the Close button to close the Vector Database Manager. Setting Trimming Parameters 2 Now, set the parameters to use the vectors added above to Click the Import button to open the Vector Import dialog. Figure : Vector Import Dialog conduct trimming. In the Trimming Parameters dialog, check the box next to the Trim Vector option. In the Select Vector list, check the TutorialVectorA box and in the Cloning Sites list, check the PrimerA box. Figure : Checkmarks for Vector and Cloning Sites From the Tutorial Data (initially located at C:\HSK_DB_Trial\TutorialData) open ContigManager / TutorialData1-TutorialVector. Choose the TutorialVectorA.prm file and click the Open button. The screen reverts to the Vector Database Manager, and TutorialVectorA will be added atop the vector list. Perform the same steps for the files TutorialVectorB.prm and TutorialVectorC.prm, to add a total of three vectors. Note that to register the actual vector to perform Similarly, check the boxes next to TutorialVectorB and PrimerB, TutorialVectorC and PrimerC, respectively. To conduct an actual analysis, check the box next to the cloning site corresponding to the imported vector. This tutorial uses a stricter value than the default for the minimum matching percentage considered as contamination, so set this to 85%. trimming on analysis data, you need to add the desired 49

51 Click the OK button to close the Trimming Parameters dialog. Return to the main window, and check the Trimming option in the navigation toolbar. The trimming results and linking direction information for each fragment will be added to the list view. Figure : List View with Additional Columns The figure above shows that fragment has a Conducting an Auto Assemble Now, perform base calls, trimming, and assembly on the trace data. First, choose all the fragments displayed in the list view in the Contig Manager main window. (To select all, click on the desired data in the list view, and then hit Ctrl-A, or choose Edit / Select All from the menu.) When all the fragments are highlighted, click the Auto Assemble option in the navigation toolbar. In this trial version, conducting an assembly will alert you of the days remaining before the Phred/Phrap option expires. After confirming, click the OK button. Figure : Remaining Trial Period Dates Display length of 927bp after trimming, with a trimming at 24bp, with TutorialVectorA as the vector used in trimming; the QV value has been established as Low, within a base number range of 379bp; and that a basecall using Phred and trimming have been conducted, and linked with normal linkage. Displaying Contig Results To display fragment data from the contigs created, choose the desired contig from the tree view in the main window. The fragments comprising the contig selected in the list view, and their respective linking directions, will display. Figure : View of All Fragments Comprising a Contig In this case, if the Phred option has been selected in the Basecall group, a basecall linking based on Phred with QV values taken into account will be conducted. If is not selected, the trace data will be linked using original basecall without Double-click on Contig1 in the tree view. The Contig Viewer will launch, and all linking conditions for Contig12 fragments are graphically shown. using QV values. When analysis is complete, the contig sequence data will be added to the tree view. Figure : Contigs Created 50

52 Figure : Graphical Display of Linking Conditions Figure : Trace Data Displayed in Parallel Map view Sequence view The Contig Viewer screen is divided into an upper and lower section. The top section includes a comprehensive graphic map view. The lower section, the sequence view, shows the highest contig sequence along with each fragment sequence. The display condition for trace data will change according to the chosen sequence position in the Contig Viewer. Click on a contig sequence in Contig Viewer or on a base atop the fragment sequence to display all trace data with the chosen basepair at the center. Displaying Trace Data in Parallel To evaluate assembled results, it is possible to view multiple trace data, in parallel. Figure : Corresponding Display when Clicking on a Basepair atop the Contig From the Contig sequence, choose the trace data to align and display, and highlight the basepair desired. Then, click the View Chromatogram option from the menu, or click the Show Chromatograms button ( ). Figure : Choosing Basepairs Figure : Corresponding Display when Clicking on a Basepair atop the Fragment Trace data will be displayed, with the selected basepair at the center. Reassembly Now, reassemble with the newly added sequences and fragments. 51 From the Start menu, choose Program / DNASIS

53 MAX Trial V2.0 / Tutorial Data to open the TutorialData folder. Then, open the folder ContigManager-TutorialData1 -TraceData1_2. Drag and drop the data within this folder to the Root folder in the tree view. The import summary dialog will display the entered sequence name and instances. Click the OK button to close the dialog. From the tree view, select the Root folder. The appropriate fragment will have been added to the list view. To conduct a basecall using Phred, and to calculate the QV value, choose the corresponding fragment and click Basecall from the navigation toolbar. To conduct vector trimming, choose the corresponding fragment and click the Trim button in the navigation toolbar. If you choose all the fragments in the list view and click the Assemble button, a message, shown in Figure , will appear. Click Yes to continue the procedure. Figure : Confirmation Dialog Using the added fragments, the reassembly process newly generates a contig sequence. If you used the sample tutorial data, the new sequence will be linked to one contig. Closing Contig Manager To close Contig Manager, choose File / Exit from the menu. 52

54 Using Quality Values for Analysis This section describes using quality values calculated from Phred as a basis for choosing sequences, as well as conducting homology searches limited to high value quality Setting Sequence Masking Parameters Double-click on Phred Quality Value Masking within the space to open the Phred QV Masking Parameter Editor. Figure Phred QV Masking Parameters Editor ranges. Note that for this tutorial, you need the DNASIS MAX DNASpace option installed. Launching DNASpace Opening a Space From the Start menu, choose Program / DNASIS MAX Trial V2.0 / DNASpace Trial. The Open Space dialog will appear, as shown in Figure Figure 8-2-1: Open Space Dialog For this tutorial, each end QV value moving average is 29, the entire sequence moving average is 19, and the high QV bp to be output to the NG folder is set as less than or equal to 500bp. Figure 8-2-4: Masking Parameters From the list, choose the option, A-09. Blast Search Acknowledging Phred QV Values and click the Open button. The space shown in Figure will open. Figure 8-2-2: A-09. Blast Search Acknowledging Phred QV Values Figure 8-2-5: Output Parameters Setting Up Trimming This tutorial uses the vector registered in an earlier section of this manual, entitled Linking Trace Data. If the vector you want to trim is not yet registered, refer to the section entitled Adding Vector Data before continuing with this tutorial. 53

55 Double-click on the option, ***** Double-Click and Set Up Figure 8-2-8: Blast Search Parameters Vector ***** to open the Trimming Settings dialog. At the top section, put a check in the box next to Trim End. Set the 5 END values to trim the first 10 bp, while the quality is less than 90%, and the 3 END values as the same as 5 END. Figure 8-2-6: Setting Up Trimming 1 Check targeted databases Entering Trace Data In the center of the dialog, mark a check next to the option, Trim Vector. From the Vector Name list, choose TutorialVectorA, and from the Cloning site list, choose PrimerA. Figure 8-2-7: Setting Up Trimming From the Start menu, choose Program / DNASIS MAX Trial V2.0 / Tutorial Data to open the TutorialData folder. Here, open the ContigManager-TutorialData1-TraceData1_1 folder. Choose all files in this folder, and drag and drop it to the ***** Drop trace data here ***** folder. Figure 8-2-9: Entering Sequence Data Once setup is complete, click the OK button to close the dialog. Setting Up Blast Searches Double-click on the ***** Double click this to set database Drag&Drop ***** in the space to display the Parameter Settings Dialog, shown in Figure For the purposes of this tutorial, you do not need to change the parameters. If you are conducting an actual analysis, use this dialog to set up the search target database. A dialog showing the execution progress will appear. When complete, the dialog will close. Figure : Progress Dialog 54

56 Understanding Execution Results When execution is complete, each folder in the space contains data. The entered fragment data is saved in the folder named ***** Drop trace data here *****. Figure : Confirming Data Within the Space includes those vector sequences that have been eliminated via trimming. For this tutorial data, you can see that from 30 entry datasets, 17 have had vector trimming conducted, with the top QV value replaced with N. Figure : Trimming Results Data list in the selected folder Double-click the desired data from the selected folder data list to view the corresponding data contents. Figure : Trace Data Entered The folder entitled ***** No trimmed sequence ***** includes sequences that could not eliminate vector sequences. For this tutorial data, from 30 entry datasets, 13 are classified as being unable to conduct vector trimming. The folder entitled ***** High quality region is long. ***** includes the data that, as a result of Phred QV Masking, contains an upper QV range bp that is longer than the default length (500bp in this tutorial). In this tutorial, out of the 17 datasets which had trimming conducted, 12 have been determined to have longer QV ranges. Double-click on the data to view results: The low QV rang value masked with N. The folder entitled ***** Basecall result by Phred ***** includes Phred basecall data. You can double-click the data to view the trace data, along with the original Basecall, Phred Basecall and Phred QV value. Figure : Masking Results Using Phred QV Figure : Phred Results Original Basecall Phred Basecall Phred QV Value Trace Data The folder entitled ***** High quality region is short. ***** includes the data that contains an upper QV range bp The folder entitled ***** Trimmed sequence ***** that is shorter than the default. In this tutorial, out of the 17 55

57 datasets which had trimming conducted, 5 have been determined to have shorter QV ranges. The folder entitled ***** Blast Search Results ***** contains Blast search results on data with vector sequences eliminated, with a QV top range of 500bp or longer, and the lower range masked by N. You can double-click on the data to view a graphic display of the results. Figure : Blast Search Results 56