De novo genome assembly

Transcription

1 BioNumerics Tutorial: De novo genome assembly 1 Aims This tutorial describes a de novo assembly of a Staphylococcus aureus genome, using single-end and pairedend reads generated by an Illumina R Genome Analyzer II. It illustrates how to build an assembly pipeline by combining a number of predefined actions. Furthermore, it will be illustrated how to change the project pipeline by inserting and removing actions, how to create user-defined action and project templates, how to export project reports and clean up project data after finalizing the project. 2 Example data The sample data set is available from the Applied Maths website ( download/sample-data, click on Power Assembler de novo project). Simply download the ZIP file and unzip the sample data to a destination folder of your choice. The Power Assembler de novo project data set contains three FASTQ data files (SRR088897, SRR and SRR ), containing 350 single-end and 881,641 paired-end reads from Staphylococcus aureus strain MRSA177. This data set is a randomized subset of sequencing run SRR The latter was downloaded from the NCBI Sequence Read Archive ( and converted to FASTQ files with the SRA toolkit ( software). In order to run this tutorial successfully, we recommend using a computer with at least 4 GB RAM memory. 3 Creating a new power assembly project 1. Create a new database (see tutorial Creating a new database ) or open an existing database. When creating a new database, it is recommended to use a SQLite or SQL Server (Express) relational database instead of a MS Access database as MS Access has performance issues when saving the assembled sequence to the database, especially when the sequence coverage is included. If no suitable sequence experiment type is present in the database, a new one needs to be created: 2. Click in the Experiment types panel to activate this panel and select Edit > Create new object... ( ). 3. From the Create a new experiment type dialog box that appears, highlight Sequence type and press <OK>. 4. Enter e.g. PA genome sequence as Sequence type name and press <Next>. 5. Leave Nucleic acid sequences selected and press <Finish> to create the new sequence type. Next, we can create a new Power Assembly project: 6. Click the tab of the Power assemblies panel from the Main window.

2 2 7. Select Edit > Create new object... ( ). 8. Enter a project name (e.g. Demo de novo ) in the Create new power assembly dialog box and press <OK>. The Demo de novo power assembly is now listed in the Power assemblies panel and will be opened in the Power assembly window. 4 Constructing and executing a de novo assembly pipeline 4.1 Importing the sequence reads We will start the tutorial for de novo genome assembly from an empty project pipeline. The analysis pipeline will be constructed by adding actions one by one. This in contrast to the tutorial on resequencing with the Power Assembler, where the use of predefined project templates is illustrated. The first action needed for this project is the action to import the FASTQ-formatted sequence reads. 1. Add the first predefined action to the empty project pipeline by selecting Action > Add action... ( ). This will open the Load action dialog box (see Figure 1). Figure 1: The Load action dialog box, with the action Import reads from FASTQ file highlighted. 2. Select the action Import reads from FASTQ file from the Import folder under Predefined actions and press <OK> to add the action to the project pipeline. The action Import reads from FASTQ file is now added to project pipeline and the Report panel displays the action-related information. The action s status is indicated as To be calculated. By using this action, we will import the three FASTQ files containing the sequence reads. By default, each read file is imported as coming from a separate sample. If multiple read files contain data from a single sample, then these samples (i.e. read files) need to be merged. In this example, the reads are

3 4. Constructing and executing a de novo assembly pipeline 3 contained in three different files, so we do need to run the action Merge samples after the import action. 3. Select Action > Add action... ( ) and add the action Merge samples from the Preprocessing folder to the project pipeline. By default, no intermediate data is saved when an action is executed. This implies that each time recalculation is needed, the project will start calculating from the beginning. To avoid this, we can define for any individual action whether or not to save the data set once the action has been calculated. When intermediate data is saved, a floppy disk icon ( ) is displayed in the upper right corner of the action. For the actions we just added to the pipeline, no intermediate data is saved so no floppy disk icon is currently shown. We will change this for the action Merge samples. 4. Highlight the action Merge samples in the project pipeline and select Action > Properties... ( ). This will open the Action properties dialog box for this action (see Figure 2). Figure 2: The Action properties dialog box for the action Merge samples. 5. Check the option Save intermediate data and press <OK>. The Merge samples action block in the project pipeline now displays the floppy disk icon, indicating that the intermediate data of the action will be saved. We will now execute the actions to import the reads and to merge them in one data set. 6. Highlight the first action Import reads from FASTQ file. In the Report panel, now the action-related report is updated. 7. Now, the existing pipeline can be ran at once by selecting Action > Execute from current ( ). This command is used to run a number of actions successively, starting from the selected action. The Import reads from FASTQ file dialog box (see Figure 3) is now displayed, asking for the runtime operator parameters. 8. Under Files to import, press <Browse> and browse for the location of the downloaded S. aureus FASTQ files (see 2). Select the three FASTQ files from the folder Power Assembler de novo project and press <Open>. Alternatively, drag and drop the files in the dialog box. The three files are now added to the Files to import list. The file SRR fastq contains the single-end reads, whereas the files SRR fastq and SRR fastq contain the paired-end reads. 9. Check Import as paired-end reads to let the software search for files that contain paired-end reads. These files should have the same name except for the last two characters, which should be 1 and 2. In this example, files SRR fastq and SRR fastq will be imported as paired-end reads. Press <Next> to continue. By pressing <Next>, the dialog pages of the next action(s) will be displayed. When pressing <Next> on the last dialog page of the project, the calculation is automatically started. In this case, as the Merge samples action has no runtime parameters defined, no second dialog page is displayed and the calculation of both actions is automatically started.

4 4 Figure 3: The Import reads from FASTQ file dialog box. The progress of the calculation is indicated with a green progress bar, displayed in the action boxes in the project pipeline. When the execution is completed, the action s status has changed to Completed and the action s reports are updated. 10. Select the action Import reads from FASTQ file from the Project pipeline panel to display the report in the Report panel. The report shows that three files were imported which contained 350 single-end reads and 881,641 paired-end reads. 11. Again, select the action Merge samples to display the action report. From this report it can be seen that all samples were merged into one new sample record. Next, we will add an action to calculate the global read statistics. 12. Add the action to the project pipeline by selecting Action > Add action... ( ). Select the action Global read statistics from the Statistics folder under Predefined actions, and press <OK> to add the action to the project pipeline. 13. Highlight the action Global read statistics and select Action > Execute ( ). The calculation of the action starts immediately, without asking for parameters. After execution, the status of the action has changed to Completed and the report is updated. The report shows statistics that contain information on the sequence length, the quality of the sequence reads and the base frequencies. From the base frequencies table, it can be seen that a number of N s are present in the data set, which will be filtered later by the trimming action. Additionally, histograms of the read sequence length, the minimum read

5 4. Constructing and executing a de novo assembly pipeline 5 quality and the average read quality are created. These summary graphs are displayed in the report. 14. To open e.g. the summary graph of the read sequence length in the Summary graph panel, right-click the graph in the report and select Open in dedicated window. The Summary graph panel is now shown with the summary graph report below. 4.2 Quality trimming An important step in the assembly pipeline is the removal of reads and parts thereof that do not fulfill the quality criteria. For this, we need to add an action that performs the quality trimming. 15. Select Action > Add action... ( ). Next, highlight the action Quality trimming (automatic) from the Trimming folder under Predefined actions, check the option to save the intermediate data and press <OK> to add the action to the project pipeline. 16. Highlight the newly added action and select Action > Execute ( ). In the Quality trimming (automatic) dialog box that appears (see Figure 4), the Minimum quality of a read and the Minimum quality of an individual base are questioned. Based on these two values, different trimming parameter values are calculated. Figure 4: The Quality trimming (automatic) dialog box. 17. Leave the default values and press <Next> to start the calculation of the action. Once finished, the action report is updated. Note that the action Global read statistics is ran again when the action Quality trimming (automatic) is executed. This is necessary because no intermediate data were saved for the former action (no icon displayed for the action Global read statistics). 18. Click the tab of the Report panel to return to this panel. In the report, the automatically determined parameter values to remove reads with low minimum or average quality, to remove low quality tails of the reads, or even to replace low quality bases in the reads, are displayed. Next, the results of the trimming actions are shown in the report. Additionally, a series of summary graphs were created. We will look in detail to two of these summary graphs, one summary graph where the trimming parameter is determined by an expression, and one summary graph where the parameter

6 6 value can be changed graphically. 19. In the Action data panel, right-click the graph Average read quality (before trimming) and select. The graph is now displayed in the Summary graph panel (see Figure 5). Figure 5: The Average read quality histogram (before trimming), as displayed in the Summary graph panel. 20. Optionally, use Display > Summary graphs > Zoom to fit ( ) to automatically fit the graph to the window size. From this representation, the lower value threshold for the parameter is visualized by the burgundy line and the shading (see Figure 5). The burgundy color indicates that this parameter is determined by an expression and cannot be changed graphically. In contrast, if the graph Read length (after tail removal) is displayed in the Summary graph panel, a red line indicates the trimming threshold. 21. In the Action data panel, right-click the summary graph Read length (after tail removal) and select. When navigating over the red line, the mouse cursor changes into a horizontal two-headed arrow, which allows to drag and drop the threshold at any value in the summary graph. From the moment a new parameter value has been changed graphically, the status of the action automatically changes to To be calculated. 22. In the Summary graph panel, drag the red line to a position corresponding to e.g. 92 bases. This way, a new value for the minimum read length is graphically defined. 23. As the action needs to be recalculated with the updated parameter value, highlight the action Quality trimming (automatic) from the project pipeline and select Action > Execute ( ) and press <Next>. 4.3 The actual assembly Once the trimming is finished, we can proceed with the actual de novo assembly, performed by the action Create de novo target (Velvet). This action uses the Velvet tool [?] to generate the de novo contigs, with a minimum contig length of 300 bp, and for each of the contigs, an assembly view will be created. The second action Determine covered regions will process only that part of the de novo assembly that has a sufficiently high coverage, and the last action Export single sequence to database will concatenate all contig sequences to a single sequence which is then exported to the database.

7 4. Constructing and executing a de novo assembly pipeline 7 First, we need to add the Create de novo target (Velvet) action to the project pipeline. 24. Select Action > Add action... ( ) to open the Load action dialog box and highlight the action Create de novo target (Velvet) from the De novo assembly folder under Predefined actions. Check the option to Save intermediate data and press <OK> to add the action to the project pipeline. In this project we imported paired-end data, typically used to help order and orient contigs from a sequencing project, this is also referred to as scaffolding. Further, paired-end reads significantly improve de novo assemblies by completely or partially spanning highly repetitive genomic regions, needed to identify structural variation. The option to perform scaffolding is by default disabled in the action Create de novo target (Velvet). However, for this example, we will enable this option as we want to generate an assembly with longer contigs and fewer, larger scaffolds. This parameter can be changed in the design of the action. 25. Highlight the action Create de novo target (Velvet) from the project pipeline. 26. Click the tab of the Action design panel. This panel gives an overview of the existing operators, and the action flowchart of the selected action. This panel is used to build or change an action or one of its parameter settings. The option to perform scaffolding is present in the Velvet tool, and as such can be defined in the operator De novo assembly (Velvet) which is present as the second operator in the action flowchart. 27. Double-click the purple operator box De novo assembly (Velvet) to open the De novo assembly (Velvet) dialog box (see Figure 6). This dialog box contains all default Velvet parameters, separated over a number of dialog tabs. From this dialog, the single-end and paired-end libraries can be defined and specific algorithm details can be entered. One of these algorithm details is the option to perform scaffolding. 28. Select the tab Algorithm details. From this tab, the k-mer length, the Minimum contig length, the option to Perform scaffolding and the option to Perform strand-specific assembly can be set. 29. Check the option Perform scaffolding and leave all other settings at their default values. Press <OK> to close the De novo assembly (Velvet) dialog box. Once this settings is defined, the remaining actions can be added to the project pipeline. 30. Repeat Instruction 24 for the action Determine covered regions from the Postprocessing folder and the action Export single sequence to database from the Export folder. For these actions, no intermediate data should be saved. After the actions Determine covered regions and Export single sequence to database are added to the pipeline, these last three added actions can be executed. 31. Highlight the action Create de novo target (Velvet), which is the action where the calculation should start, and select Action > Execute from current ( ). 32. In the Runtime parameters dialog box, the first page Read libraries for de novo assembly is displayed (see Figure 7). Check the option to use the paired-end reads in the data set, leave all other setting to their default values and press <Next>. 33. Leave all settings on the following pages to their default values and press <Next> until the page Export single sequence is displayed (see Figure 8). 34. Enter e.g. denovo S.aureus for the Entry key and select the sequence experiment where the sequence should be saved to from the drop-down list (see Figure 8). Press <Finish>. The calculation of the highlighted action (Create de novo target (Velvet)) is now started and the remaining actions (Determine covered regions and Export single sequence to database) are executed one by one. As a result, the concatenated de novo contigs will be exported to the database. Depending on your system,

8 8 Figure 6: The De novo assembly (Velvet) dialog box. the computation time for this de novo analysis may take some minutes. The progress of the calculation is displayed in real-time in the Execution log panel. To display the Execution log panel, simply click its corresponding tab. As can be seen from the project, this log file contains detailed information on the start and end time, the time elapsed and a descriptive message for each operator. Additionally, the information provided by third-party tools, e.g. in this case the Velvet program, is streamed to the execution log of the action. After the calculation of the last action has finished, we can return to the project pipeline again. 35. Select the action Create de novo target (Velvet) and display the Report panel by clicking on the corresponding tab. From this report, it can be seen that both single-end and paired-end data is used for the assembly. Furthermore, all the different parameters used by Velvet are indicated. Notice that the paired-end information was used to improve the alignment. 36. In the Create de novo target (Velvet) action report, click on the minus sign next to Parameters to collapse the parameter information.

9 4. Constructing and executing a de novo assembly pipeline 9 Figure 7: The dialog page Read libraries for de novo assembly of the Runtime parameters dialog box for the action Create de novo target (Velvet). Figure 8: The dialog page Export single sequence of the Runtime parameters dialog box for the action Export single sequence to database. When we look at the results, we can see that this assembly resulted in 287 sequences (contig length > 300 bp) which were added to the data set. Additional to the formation of the de novo contigs, an assembly was created on these contigs to map the reads back to the de novo sequences. Final alignment of the reads on the contigs results in about 89,8% of the reads that are mapped. 37. Click on the minus sign next to Map reads to target (Velvet) to collapse this information.

10 10 Subsequently, the information of the coverage and consensus base calling is displayed. 38. Again, click on the minus signs to collapse these parts in the report. Finally, for each contig, the action/pipeline (used with default settings) will create an assembly view, a sequence curve, a sequence quality curve, a sequence coverage curve and a quality matrix curve. If the action that created the de novo contigs is highlighted in the project pipeline, these assemblies and sequence curves can be loaded to the Assembly panel and Sequence curves panel, respectively. 39. Highlight the action Determine covered regions or Export single sequence to database to update the corresponding reports. The latter action report indicates the Entry key and the Sequence experiment, where the Power Assembler output sequence is exported. 40. To verify that this last action indeed completed, return to the Main window and click on the colored dot for key denovo S.aureus and sequence type PA genome sequence. The Sequence editor window appears with the target sequence that was exported from the Power Assembly project. This ends the actual de novo assembly pipeline. The following parts of the tutorial illustrate how to manipulate the project pipeline, how to create user-defined actions and project templates, and how to clean up project data. 5 Side note: Anatomy of an action The logic of the Power Assembly and the structure of an action can be illustrated using the action Global read statistics. As previously stated, a power assembly pipeline consists of a sequence of actions, graphically represented in the Project pipeline panel. At a deeper level, the structure of an action is displayed in the Action design panel. 1. Select the action Global read statistics and click on the tab of the Action design panel to bring this panel into focus. The Operators panel contains an overview of all the operators, sorted by category. Any combination of operators can be used to create the action flowchart (see Figure 9), which basically represents the functionality of the action. In the Action flowchart panel, one can view the information present at the operator level. For each operator block in the action flow chart, two names are displayed. On top, the user-defined operator name is shown in bold. If no user-defined name is defined, (no name) is displayed. At the bottom, the operator name as defined in the software is displayed. This can be illustrated for the Global statistics operator, the second operator in the action flowchart (see Figure 9). 2. In the Operators panel, navigate to the Statistics category and select the first operator Global statistics. When selecting the operator from the tree, a brief description is shown at the bottom of the panel explaining that this operator works on a specified set of sequence records. We now illustrate the link to one of the operators present in the action flowchart. 3. From the action flowchart, double-click the operator Global read statistics. This displays the Global statistics dialog box (see Figure 10). From this dialog box, the General parameters and Input fields for this operator can be defined. General parameters for the operator Global read statistics include: Operator name: the name of the operator is used to refer to this operator in the report and in subsequent expressions. If no name is given, the operator cannot be referred to. The operator name is

11 5. Side note: Anatomy of an action 11 Figure 9: The action flow chart for the action Global read statistics. Figure 10: The Global statistics dialog box. displayed in bold in the operator block, present in the action flowchart. In Figure 10, the operator name is defined as Global read statistics. Restriction test: this logical test is an expression that determines whether or not a sequence record is used by the operator. Only the sequence records which pass the restriction test are used by the operator. In this case, the restriction test [Is reference]==false (see Figure 10) implies that only read sequences are used by the operator. Group-by: when set, the operator determines groups based on the field specified, and runs once for each group separately. In this tutorial, the parameter is not set (see Figure 10) as we target only one sample.

12 12 The Input fields refer to which fields hold the sequence and the sequence quality information, and are in this case defined by Sequence field and Sequence quality field. To gain further insight in the action flowchart and its operators, the operators that created the histograms will be discussed. These three operators are all based on the same operator template, Create histogram, present in the Operators panel. 4. In the Operators panel, navigate to Summary graphs and select the first operator Create histogram. With this operator highlighted in the operators tree, the operator description is updated to Creates a histogram based on a value expression. To use this operator, the only thing needed is the value expression that defines which property is used in the summary graph. This can be illustrated, e.g. by the summary graph created on the read sequence length: 5. Double-click the third operator, Create read length histogram of single end reads (statistics), from the Action flowchart panel to open the Create histogram dialog box (see Figure 11). Figure 11: The Create histogram dialog box. From this dialog box, the General parameters and Operator parameters can be defined. General parameters for the operator Create read length histogram (statistics) (Operator name and Restriction test) are the same as for the operator Global read statistics, discussed above. The Operator parameters include: Name: the name of the histogram is used to refer to the histogram in the reports and in the action data. Value expression: this numerical expression is evaluated for every sequence record and results in a value that is used as input data for the histogram. The value expression for the operator Create read length histogram (statistics) (i.e. Length([Sequence]) ), will calculate the sequence length for each sequence that passes the restriction test, i.e. the sequence length of all the read sequences will be taken into account to create the histogram. Binning properties: these properties include the bin size and the fixed range that define the graph. This part of the tutorial is limited to the illustration of the logic behind the actions and the operators. Advanced editing of the operators will not be discussed here.

13 6. Changing the project pipeline by inserting and removing actions 13 6 Changing the project pipeline by inserting and removing actions This part of the tutorial we illustrate how to insert and remove an action from the project pipeline. When adding actions to the project pipeline, by default the action is added at the rear of the pipeline. Using specific settings, one can add an action amidst other actions already present in the pipeline. In the current pipeline, we can build in an action which provides some feedback on the generated de novo assembly. As an example we will insert the action Contig statistics just after the action that generates the de novo assembly. 1. Highlight the action Determine covered regions in the project pipeline and select Action > Add action... ( ). This pops up the Load action dialog box (see Figure 12). Figure 12: The Load action dialog box. 2. In the Load action dialog box, select the action Contig statistics from the Statistics folder, check the option Add new action before the currently selected action, leave the option Save intermediate data unchecked, and press <OK>. Now the action Contig statistics is added in front of the action Determine covered regions. Note that no floppy disk icon ( ) is displayed in the action block, indicating that no data will be saved after the calculation of this action. 3. Execute the Contig statistics action by selecting Action > Execute ( ). 4. Once the action is calculated, highlight the Contig statistics action in the Project pipeline panel and select the tab corresponding to the Report panel. This report displays the calculated contig statistics, i.e. the number of contigs and the size distribution of the contigs. Additional information such as the contig quality and contig coverage, as well as the sequence identity, mapping degeneracy and discrimination is summarized in the histograms. A highlighted action can be removed from the project pipeline by selecting Action > Remove action ( ). If the action generated action data, the software will ask whether these data may also be removed.

14 14 7 Customizing a predefined action In the next part of the tutorial, we will modify a predefined action and save this modified action as a userdefined template. Templates allow users to exchange their analysis methods. Currently, the action Create de novo target (Velvet) will create an assembly view, a sequence curve, a sequence quality curve, a sequence coverage and a quality matrix curve for each de novo contig. If one is not interested in these assembly and curve information, we can modify the action that no action data will be created. Note that modifying an action is done in the Action design panel. The action consists of a specific sequence of operators that are responsible for the functionality of the action. For this reason, changing the action flowchart or the parameters of an operator is not without risk and can have great influence on the performance of the action. 1. Highlight the action Create de novo target (Velvet) in the project pipeline. From the report that is displayed, we can see which operators are responsible for the creation of the assemblies and the curves. First, the parameters are listed in the report and second, the results are listed in the order they are present in the action flowchart. The operators De novo assembly (Velvet) to Consensus base calling produce the action data, whereas the operators Create assembly view to Create quality matrix curve are responsible for the visualization of the action data. In summary, the last nine operators need to be removed. 2. Click the tab for the Action design panel to display the action flowchart. In this panel, the operators that are part of the action are displayed as purple blocks and the final data set is displayed as a green block. 3. From the action flowchart, select the operator Create assembly view, hold the Ctrl-key and select the subsequent operators. For each of the selected operators, a red box is shown. 4. Select Action > Action design > Remove currently selected operators ( ) to remove the operators at once. Confirm this action, and confirm the removal of the associated curves and assemblies by pressing <Yes> twice. As the program needs to delete all data (curves and assemblies), it can take a few seconds for the program to resume. To make clear that modifications where made to this action, we will change the action name. 5. Highlight the Create de novo target (Velvet) action in the project pipeline and select Action > Properties... ( ). In the Action properties dialog box, the action name and the description of the action can be specified. Note that it is also possible from this dialog to specify whether or not intermediate data should be saved. 6. Change the name e.g. to De novo assembly (Velvet) (without action data) and the description to This action creates a de novo target sequence using Velvet without creating contig assemblies and contig curves.. Press <OK> to save the changes. The action name is now updated. The next step is to create an action template from the action we just modified. 8 Saving and exporting action templates Analysis templates can be stored in the database to be used in other assembly projects, but can also be exported to XML files. The latter option allows users to exchange analysis templates with colleagues, and/or to apply the same template in other BioNumerics databases. Both options will be illustrated. We will first add the customized action template from previous section to the database. 1. Highlight the modified action and select Action > Store action as template... This brings up the Store action as template dialog box.

15 8. Saving and exporting action templates As we already changed the name and description of the action, we can leave the values unaltered. Press <OK> to store the template in the database. 3. To illustrate that the template is indeed stored, select Action > Add action... ( ). In the Load action dialog box, press the minus sign next to the Predefined actions folder. Once a user-defined action has been saved, the folder User-defined actions becomes available from the Action tree (see Figure 13). Currently, this folder only contains a single action, i.e. the customized de novo assembly action that was just added to the database. When selecting the action, the description is shown on the right. Figure 13: User-defined action. 4. As this is just an illustration of the action templates present in the database, simply close the dialog box by selecting <Cancel>. To remove action templates from the database, use Action > Remove action templates... This brings up the Remove action templates dialog box, from where an action template can be removed. Now we will export the analysis template to an XML file. Exchange of XML templates, either action templates or project templates, is very useful as it allows e.g. to send a complete analysis workflow by To create the XML action template, highlight the action in the project pipeline and select Action > Export action template... In the Export action as XML dialog box, the Name, the Category, the Description and the XML File name can be defined. 6. Leave the name and the description unaltered and enter De novo assembly under Category. 7. Click <Browse> to select the output XML file and select <Open>. Alternatively, just type in the filename (e.g. denovo velvet custom.xml ). Press <OK> to export the action to XML. When needed, this XML project template can be imported into the action flowchart by File > Load pipeline from template... ( ).

16 16 9 Saving and exporting project templates Similar as for action templates, one can also create project templates. In this part of the tutorial we will save the current project to a new project template. 1. Select File > Store pipeline as template... In the Save project template dialog box, the name and description of the template can be specified. By default, the name of the current power assembly project will be filled in. 2. Change the name and description according your own preferences, e.g. De novo Velvet (custom1) and This project template uses Illumina SE and PE data (FASTQ format) for de novo assembly. The de novo contigs are concatenated to one sequence which is then exported to an entry in the database. No action data are created., respectively. Press <OK> to confirm. We will now verify if the project pipeline is stored as a template. 3. Navigate to the Main window, highlight the Power assemblies panel and create a new Power Assembly project by selecting Edit > Create new object... ( ). 4. Enter the name for the new project e.g. De novo pipeline in the Create new power assembly dialog box and press <OK>. The new project is now created and automatically opens. 5. Select File > Load pipeline from template... ( ) to show the Load project template dialog box. In this dialog, the existing project templates are displayed. On top, the folder of the Example project templates is shown, and at the bottom the user-defined templates are listed (see Figure 14). Figure 14: User-defined template. 6. Select the project template De novo Velvet (custom1) from the list. Note that the project description at the right is updated. Leave all settings default. In this example, the option Clear current pipeline remains checked. If unchecked, the project template is added at the rear of the existing project pipeline. For this example there is no existing project pipeline so,

17 10. Exporting the project report 17 either way, the result will be the same. 7. Press <OK> to automatically load the project pipeline from the template. To leave this digression, you can close the present project and return to the original Power Assembly project. Saved project templates can be questioned from all projects within the same database. To exchange projects between databases or users, one can use the XML export. We will use the same project pipeline to illustrate this. 8. To export the project template as XML, select File > Export pipeline In the Export project template as XML dialog box, the Name, the Category, the Description and the File name can be defined. Enter at least a name and a description for the template and click <Browse> to select the output XML file, or type the filename e.g. denovo(custom1).xml. Press <Open> to confirm. Press <OK> to export the project. The XML template is now created. Similar as to the other templates, we can simply import this project template in a new or existing project by selecting File > Import pipeline... In the Import project template from XML dialog box, one can browse for the correct XML file and confirm by <OK>. The template will then be inserted/added to the project pipeline. 10 Exporting the project report In this part of the tutorial, we will export the Power assembly project report to an HTML file and save this file as an attachment with its corresponding entry. 1. If the Demo de novo power assembly project is not open, navigate to the Main window and open this project via Edit > Open highlighted object... (, Enter). 2. In the Power assembly window, select the vertical box with the project name from the project pipeline. The project report is now displayed in the Report panel. Note the difference between selecting an action box which will only display the action report, and selecting the project box which will display the complete project report, being a sequence of the different action reports. 3. With the project box highlighted, select File > Export report as html > Report ( ). The project report is displayed in the default browser and the location where the report is saved is indicated. Optionally, you can save this report as attachment to the entry that holds the sequence information exported from this project. Saving the HTML file as attachment to the entry can be done as follows: 4. Double-click the entry in the database where the sequence information from the demo project was exported to e.g. denovo S.aureus. This opens the Entry window (see Figure 15). 5. Click the tab of the Attachments panel to bring this panel to the front. 6. Create a new attachment by selecting File > Attachments > Add new attachment ( ). This opens the Create new attachment dialog box (see Figure 16). 7. From the Create new attachment dialog box, select Document as Attachment type and browse for the correct HTML file that contains the project report. Optionally, enter Project report Power assembler as a description and press <OK> to confirm. The HTML project file is now added as attachment to the entry and remains accessible from the database entry at any time.

18 18 Figure 15: The Entry window for the database entry with key denovo S.aureus. Figure 16: The Create new attachment dialog box. 11 Cleaning up project data To reduce the amount of disk space occupied by a project, intermediate data such as reports, data sets and assemblies can be removed once a project is finished. To illustrate this, we will remove the data sets from the demo project. First, we will open the folder where the data sets are stored. 1. Select Database > Database settings... and press <Open database directory> to open this directory in Windows Explorer. 2. Successively open the folders sourcefiles, PAssemblies and data from the database folder. In the data folder, the report files and the folders that contain the saved data sets are listed. Based on the Date modified information, one can search for the data generated by this demo project and verify the presence of the saved data sets. In BioNumerics, we will now clean up these saved data sets. 3. Open the demo project from the Main window by selecting the project from the power assembly list and selecting Edit > Open highlighted object... (, Enter).

19 11. Cleaning up project data To clean up data, select File > Cleanup... from the Power assembly window. This opens the Cleanup dialog box (see Figure 17). Figure 17: The Cleanup dialog box. In the Cleanup dialog box, one can specify which data types need to be removed. 5. For this example, uncheck Search data, Sequence curves and Summary graphs, and only leave Data sets checked. Press <OK> to confirm. As can be seen from the data folder content, the data sets from the project are now removed. According to personal preferences and available disk space, one can decide which data needs to be cleaned up after a project has been finished.