For Research Use Only. Not for use in diagnostic procedures.

Transcription

1 SMRT View Guide

2 For Research Use Only. Not for use in diagnostic procedures. P/N Copyright 2012, Pacific Biosciences of California, Inc. All rights reserved. Information in this document is subject to change without notice. Pacific Biosciences assumes no responsibility for any errors or omissions in this document. PACIFIC BIOSCIENCES DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESS, STATUTORY, IMPLIED OR OTHERWISE, INCLUDING, BUT NOT LIMITED TO, ANY WARRANTIES OF MERCHANTABILITY, SATISFACTORY QUALITY, NONINFRINGEMENT OR FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL PACIFIC BIOSCIENCES BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, PURSUANT TO ANY STATUTE, OR ON ANY OTHER BASIS FOR SPECIAL, CONSEQUENTIAL, INCIDENTAL, EXEMPLARY OR INDIRECT DAMAGES IN CONNECTION WITH (OR ARISING FROM) THIS DOCUMENT, WHETHER OR NOT FORESEEABLE AND WHETHER OR NOT PACIFIC BIOSCIENCES IS ADVISED OF THE POSSIBILITY OF SUCH DAMAGES. Certain notices, terms, conditions and/or use restrictions may pertain to your use of Pacific Biosciences products and/or third party products. Please refer to the applicable Pacific Biosciences Terms and Conditions of Sale and the applicable license terms at Trademarks: Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT and SMRTbell are trademarks of Pacific Biosciences in the United States and/or certain other countries. All other trademarks are the sole property of their respective owners. Pacific Biosciences of California, Inc Willow Road Menlo Park, CA

3 SMRT View Guide Table of Contents Introduction Pacific Biosciences Software Overview Hardware/Software Requirements Accessing SMRT View from SMRT Portal Installing SMRT View on Your Desktop Opening Data Files Directly from SMRT View What s New in SMRT View v Basic Workflow Navigating Through Your Data Linking/Unlinking the Region and Details Panels Sorting Contigs by Length Viewing a Constant Visible Range Showing/Hiding Reads Navigating Using Numeric Genomic Locations Viewing Resequencing Data Viewing de novo Assembly Data Viewing Base Modifications Viewing Insertions Adding Annotation Tracks Preparing Annotation Tracks to Add to SMRT View Exporting Data Window Management Saving and Opening Layouts Menu Commands File Menu View Menu Layout Menu Tools Menu Tools Menu, Preferences Help Menu Right-Click Commands Keyboard Commands Toolbar Commands Additional Panels Selection Panel Table Browser Motif Summary Panel Troubleshooting Page 1

4 Introduction SMRT View is a whole genome browser that visualizes secondary analysis data generated by SMRT Portal, using data generated by the PacBio RS instrument. SMRT View is part of SMRT Analysis, which is available from the PacBio Developer s Network, at SMRT-Analysis. Secondary analysis includes: Aligning a group of reads to a reference sequence to produce a consensus sequence. Assembling a set of reads into contigs to produce a de novo sequence. Identifying insertions, deletions, and SNPs. Evaluating consensus quality and quality of the instrument run. Pacific Biosciences Software Overview Pacific Biosciences includes four software packages with the PacBio RS instrument: RS Remote Use RS Remote to create runs that you select from the instrument. You also use RS Remote to monitor instrument runs. When you create a run, you can specify that primary analysis data is fed directly into a secondary analysis job. RS Touch Use RS Touch from the instrument touchscreen to select a run, load the instrument, and then start the run. SMRT Portal Use SMRT Portal to create and run secondary analysis jobs using primary analysis data generated by the instrument. SMRT View Use SMRT View (a whole genome browser) to visualize secondary analysis data generated by SMRT Portal, using primary analysis data generated by the instrument. Page 2

5 Hardware/Software Requirements Hardware We recommend: A minimum screen resolution of 1280 by 800 pixels. A minimum of 4 GB of RAM. Software SMRT View will run on the following: Operating Systems Microsoft Windows XP, Microsoft Windows 7 (32 bit and 64 bit) Mac OS X 10.5 or later Java Version 1.6.0_14 or later (Windows) 1.6.0_17 (Mac OS X) Accessing SMRT View from SMRT Portal Use this procedure to visualize a specific secondary analysis job from SMRT Portal. 1. Click the View Data tab to see a list of all completed secondary analysis jobs. 2. Select a job, then either: Click SMRT View at the bottom of the screen, or Click Open at the bottom of the screen to view the Reports page for the job, then click SMRT View. Installing SMRT View on Your Desktop You can install on your local Windows/Macintosh/Linux computer a desktop shortcut to the version of SMRT View on the remote server, where SMRT Analysis is installed. You can install multiple shortcuts if you have access to multiple SMRT Analysis servers. There are two ways to install the shortcut: From within SMRT View: Choose Tools > Install SMRT View, or Access the SMRT Analysis Server page, click SMRT View Home Page, then click the Here link. (The page also includes information on how to remove SMRT View from your desktop.) Starting SMRT View From Your Desktop 1. Double-click the desktop shortcut. This launches SMRT View in a limited mode where you can only choose File > Open Data from Server. (See below for the file types that you can open.) Page 3

6 Opening Data Files Directly from SMRT View 1. Choose File > Open Data from Server. You can open three types of files located on the SMRT Analysis server: Job Metadata (.rdf) This file is generated when you create a job using SMRT Pipe, and is located in a Jobs folder. The.rdf file is associated with a specific Reference Metadata file (reference.info.xml). To specify a different reference file, click Reference and choose a different file. Aligned Reads (cmp.h5) This file is generated when you create a job using SMRT Pipe, and is located in a Data folder. After selecting a cmp.h5 file, you must select a reference.info.xml file (located in a References folder). Reference Metadata (reference.info.xml) This file is imported into SMRT Analysis, and is located in a References folder. The file describes the reference and its contigs, and also contains information about its associated data files, such as FASTA sequence and annotations. 2. Click OK. The data displays in SMRT View. What s New in SMRT View v Supports motif visualization If the data was generated using the RS_Modification_and_Motif_Analysis protocol. Displays one track per motif detected: Displays the Motifs Summary panel: 2. Supports the identification of the following base modifications: N6-methyladenine (m-6a) 4-methylcytosine (m-4c) 5-methylcytosine (m-5c) when converted with TET. Page 4

7 3. You can now filter the data displayed in the Region and Details panels using the Table Browser. 4. Updated navigation options: Relationship between multiple panels is simplified. You can now lock the visual range in the Region panel: New option to specify whether or not to display subreads in the Details panel. Not displaying the reads results in faster performance. (This option may be turned on or off by default depending on the type of job visualized.) Simplified toolbar: New Details panel Coverage Limit option to limit maximum coverage; a lower limit increases performance. Page 5

8 Basic Workflow SMRT View displays secondary analysis data using three different panels: The Genome panel, Region panel, and the Details panel. Navigating Through Your Data Linking/Unlinking the Region and Details Panels 1. (Optional) In the Genome panel, click a genome, chromosome or DNA segment to select it. Or, click and drag to select a section of interest. The Region panel displays the selection in greater detail. 2. In the Region panel, click and drag to select a smaller section. That section displays in the Details panel. 3. In the Details panel, click and drag to view the smallest area, down to the individual bases. You can quickly unlink the three panels so that they display data independently of the other panels. Unlinking makes it easier to navigate and explore your data as needed. It also can make the application faster as you do not have to load in as much data. You can easily link the panels as needed so that they are back in synch. Note: When you select a different contig, the Region and Details panels always display that contig, whether or not the panels are linked. Page 6

9 To link/unlink the Genome and Region panels, or the Region and Details panels: Click the Link/Unlink control in the Region or Details panel toolbar. You can also edit the Global Preferences to link or unlink panels. The Genome panel: Displays whole chromosomes or DNA segments, along with significant points of interest. Displays only if the secondary analysis data includes multiple genomes, chromosomes, or segments. Sorting Contigs by Length Click the Sort icon in the Genome panel toolbar. (Note: This setting is not saved when you exit SMRT View.) The Region panel: Acts as a summary of the data. Displays metrics such as coverage and variants, and allows you to quickly navigate across your data to identify regions of interest. Page 7

10 Viewing a Constant Visible Range When you select an area in the Region panel, you can specify that it defaults to the same visible range. This displays the same size range in the Details panel, making navigation easier. Click the Visible Range control in the toolbar: The Details panel: Allows you to drill down to base-level resolution and visualize SNPs and base modifications. Displays a pileup of the reads. Displays different tracks, depending on the experimental data viewed. (A resequencing experiment displays variants tracks, while a base modification experiment displays both variants and template tracks.) Displays the reference sequence. (Right-click and choose Zoom to Base to see the individual bases.) Note: The colors used for the reference sequence bases are different from that of any annotations. Displays the direction (forward or reverse) of read strands. Displays base modifications. (Choose Tools > Preferences, click Base Modifications, check Show Kinetogram and Base Modification annotations, then select the type of metric to view.) See Viewing Base Modifications on page 13 for further details. Displays insertions. (Select a region, then right-click and choose View Insertions.) Page 8

11 Showing/Hiding Reads For better performance, you can choose to not display the reads in the Details panel: Click the Show Reads/Hide Reads control in the toolbar. Note: There are some significant display differences if you are visualizing data from a de novo assembly. See Viewing de novo Assembly Data on page 11. Navigating Using Numeric Genomic Locations This method is useful if you already know the numeric location of the genomic region to display. 1. Choose Tools > Go To Location, or click the Go To arrow on the toolbar. Page 9

12 2. If the data includes multiple genomes, chromosomes, or segments, select a contig from the Contig list. 3. Go to a specific location in the Genome panel: Enter a starting position and an ending position, then click the >> button. 4. (Optional) Go to a specific location in the Region or Details panel: Enter starting and ending positions, then click the >> button. (Note: These fields are available only if the Region and/or Details panels are unlinked.) In any of the 3 panels: Viewing Resequencing Data Move the mouse over areas of interest to display tool tips with additional information. (To specify whether or not to display tool tips on the Details panel, choose Tools > Preferences and check or uncheck Show Tooltips for Reads.) Use the + (Plus) key to zoom in and the - (Minus) key to zoom out. Use the Left and Right arrow keys to scroll through the genomic range. When viewing resequencing data, you typically look to verify the quality of your data, verify the variants, and then validate any issues. The Details panel displays variant tracks, which show any differences between the reference track and the reads for both strands. Specifying Variant Annotation Colors Specifying Variant Annotation You can specify the colors associated with individual DNA bases used to display substitutions, deletions, insertions and normal annotations. This can help you more easily view the annotations in the Details panel. 1. Choose Tools > Preferences, then click Variants Annotations. 2. Click the color next to the desired annotation type. 3. Choose a different color and click OK. 4. Click OK. The change takes effect immediately. 1. Choose Tools > Preferences. 2. Click Variants Annotations. 3. Check Filter Variants by Coverage. 4. Enter a number between 1 and 100. Variants are filtered based on the number that you enter. The higher the number, the higher the coverage must be to view the variant. (This setting affects all panels.) 5. Check Filter Base Annotations by Variant Event. 6. Enter a number between 1 and 10. Annotations are not highlighted unless they occur at the same genomic location of a variant where the coverage is at least a number that you entered. (This setting affects the Details panel only.) 7. Click OK. The changes take effect immediately. Page 10

13 Specifying Quality Value Annotations 1. Choose Tools > Preferences. 2. Click QV Annotations. 3. Check Show Base QV Annotations. 4. Enter QV values in the Threshold column. Quality values are used to change the color intensity of annotations based on the numbers that you entered. Base QV thresholds are on a per-read and perbase basis, and come from primary analysis. Not all files include QV thresholds, so this option may not be applicable in all cases. 5. Check Show Map QV Annotations. This option changes the color of reads to display the calculated Phred-scale read map quality value. This value estimates the probability that a read is incorrectly mapped to its displayed mapped location. 6. Click OK. The changes take effect immediately. Notes: Viewing de novo Assembly Data The colors displayed for Show Base QV Annotations are derived from the colors you selected for the Variants Annotations. These settings affects the Details panel only. Following are display differences if you are visualizing data from a de novo assembly: The full de novo assembly is shown in two tabs: The De Novo Assembly tab, and the De Novo Assembly Summary tab. The Region and Details panels display detailed information about the selected contig. The top coverage contig is usually the control sequence, with other peaks being repeat sequences. If you zoom in the Details panel, you see the de novo consensus, which uses the same track used for the reference in resequencing. In the Details panel, SNPs are not shown in a separate track. The total number of variations is summarized in the Region panel's variations track (assembled_summary.gff.gz). Page 11

14 You can also view contig information in a table format by clicking the DeNovo Assembly Summary tab: To display data from a specific contig in the Region and Details panel: Click a table row. To sort data: Click a column header to sort the table on that column. Page 12

15 Viewing Base Modifications Base modification data can be helpful in visualizing DNA modification, such as methylation. SMRT View can identify and display the following base modification events: N6-methyladenine (m-6a) 4-methylcytosine (m-4c) 5-methylcytosine (m-5c) when converted with TET Note: Full base modification visualization is possible only if you set up the secondary analysis job in SMRT Portal using: Pacific Biosciences' RS_Modification_and_Motif_Analysis or RS_Modification_Detection protocol, or Your own protocol which includes the base modification module. Base modification jobs include several additional visualization tracks: 1. The Summary track displays the different types of base modifications in a stacked histogram. The track is colored both by strand and by the type of modification. 2. The Modification Events track displays modification events for both the plus and minus strands, colored by strand. Each "event" detected by the analysis displays as a color-coded marker. The label modified_base refers to any potential modifications that are not specifically called out by the software, but still pass basic statistical thresholds to flag that location for further analysis. The Modification Events track displays in both the Region and Details panels. Only events with a confidence value above the user-selected threshold display. To change the threshold value, choose Tools > Preferences, click Base Modifications, then enter a value in the Filter Modifications Events by Confidence field. The SMRT View display differs based on the protocol used to produce the data: RS_Modification_Detection protocol: Displays one track per modification type detected; no motifs are displayed. Modifications that do not fall within the bounds of a methyltransferase motif are all Page 13

16 placed in a separate track labeled others. (Data is read from the modifications.gff file.) Following is the Region panel display: Details panel display: RS_Modification_and_Motif_Analysis protocol: Displays one track per motif detected. (Data is read from the motifs.gff file.) Region panel display: Details panel display: 3. The Kinetogram displays in the Details panel, for each position for each strand, next to the reference sequence view with both strands indicated. Note: The kinetogram is visible in the Details panel only when zoomed in to single base level. The reference sequence in the Details panel is split by strand, and a normalized IPD is shown for every position in the alignment, displayed as colored bars. Pulse width is never shown in the kinetogram. Page 14

17 4. The reference sequence is separated for each strand and shown in template space (the base displayed is what was in your original template molecule) with a histogram representing the normalized IPD (inter pulse duration) at each position. Template space indicates the base in your DNA template that may be modified. For example, if an adenine base is flagged in the modifications track and has a high normalized IPD value, it may indicate a methyladenine in the template. This will correspond to an identified methyladenine if identification is activated. An IPD ratio greater than 1 means that the sequencing polymerase slowed down at this base position, relative to the control. An IPD ratio less than 1 indicates speeding up. Additionally, all sequences in the reference and for aligned reads are oriented in template space. If an A is shown as putatively modified in the display, then it was the original A that may be modified. The modification track showing significant events is labeled with a base context always in the 5'->3' direction in template space for events on both strands. 5. When View Reads (on the Details Panel title bar) is switched on, each aligned read is also shown, separated by strand, in template space. Aligned reads at the bottom of the Details panel are colored by one of three types of annotation: Variants and Base QV, Raw Interpulse Duration (in seconds), or Pulse Width (in seconds). Page 15

18 Viewing Base Modifications 1. Choose Tools > Preferences, then click Base Modifications. 2. Check Show Kinetogram and Base Modification Annotations. 3. Select one of the available types of base annotations. (You may have to wait a bit for the additional information to be retrieved from the server.) Note: Steps 4-6 do not apply if you selected Variants and Base QV. 4. Choose the type of scaling to use for the display: Linear, Log10, Square Root, or Exponential. The color display on the right changes based on the scaling you select. 5. (Optional) Specify minimum and maximum kinetic measurement values to display. The color display on the right changes based on the values you selected. 6. (Optional) Change the colors of the reverse or forward strand: Double-click the upper part (forward strand) or lower part (reverse strand) of the color bar. 7. (Optional) Check Filter Modification Events by Confidence and enter a value. Base modification events in the modifications (events) track display only if the confidence score is at least as high as this value. The higher the number, the higher the coverage must be to view the event. (This option is checked by default, with a cut-off confidence value of 50.) To see all base modification events: Uncheck Filter Modification Events by Confidence. 8. Click OK. The change takes effect immediately. Viewing Insertions When you right-click a region of data in the Details panel and choose View Insertions, SMRT View displays insertion annotations. SMRT View displays the 5 bases before and the 5 bases after the insertion. If you click an area with no insertions, SMRT View searches the first 100 bases to the right of the position you clicked to find an insertion. Page 16

19 Adding Annotation Tracks If the Show Kinetogram and Base Modifications annotations preference is checked, the dialog displays the base modification annotations. To print the contents of the dialog: Click Print. To export the contents of the dialog as a PNG or PDF file: Click Export Graphics. You can add annotations for specific organisms, from external sources, that will display in SMRT View. You can also add annotation files that you create, as long as you use one of the supported file formats. You can add annotation tracks into SMRT View in the following formats: GFF3 (Well-formatted files.) GTF (These are typically available on the UCSC Genome Browser web site.) VCF GZ (GNU Zip compressed archive file. Note: The compressed file must be in GFF3 format.) Each data file added to SMRT View is shown in an individual track. Preparing Annotation Tracks to Add to SMRT View Annotations that you add to SMRT View must be compatible with the current reference sequence, and must be written using one of the supported file formats. Reference sequence Annotation files added to SMRT View must correctly identify the reference sequence (contig) of each feature. To correctly import a GFF3/GTF/VCF file, the contig names found in the file to import must match the contig names of the corresponding reference used for that analysis. In a GFF3 file, this is the value found in the first column. Page 17

20 Contig names are also shown in SMRT View's panel headers and the Session Properties dialog. If a match is not found, SMRT View will not import the feature. Note: When you import reference sequences using SMRT Portal, a default contig name derived from the FASTA file header is assigned to each FASTA reference. All reference sequences, contig names and contig IDs used by the secondary analysis system are stored in the References Repository, located in the directory $SEYMOUR_HOME/common/references. Adding Annotation Tracks 1. Go to a source where you can find and download annotations. Some good sites include: UCSC Genome Browser (Table Browser) Ensemble Genome Browser UCSC Microbial Genome Browser UCSC Cancer Genomic Browser 2. Select and download files in GFF3, GTF, VCF, or GZ format, and save the files to your local hard drive or network share volume. 3. In SMRT View, choose either File > Add Tracks (for local files) or File > Add Tracks from Server. 4. Select the file containing the annotation tracks and click Open. 5. Annotation data now displays in the Genome/Region/Details panels, in a new track named using the name of the file. The annotations do not persist after you close SMRT View. Page 18

21 Exporting Data Exporting Graphics Exporting Data When you export graphics, all data in all panels are exported as individual PNG or PDF image files. This includes data that scrolls offscreen. 1. Choose File > Export Graphics. 2. Specify which panel(s) to export graphics from. 3. Name the file and choose a location. 4. Specify the image file type: PDF or PNG. 5. Click Save. You can export data from the Table Browser, Region panel, or Details panel in GFF3 format as input for further downstream processing, to pass on to collaborators, or upload to public genome sites. Exported data includes any Region or Details panel data that scrolls off-screen, as well as annotations, tracks, genes, variants, and all reads. 1. Choose File > Export Data. 2. Select the data source: Table Browser, Region Panel, or Details Panel. 3. Name the file and choose a location. 4. Click Save. Notes: Window Management When you filter data in the Table Browser, only the data that displays is exported. If you specify that reads not be shown in the Details panel, the reads data will not be exported. Use the window controls in each panel's title bar to minimize or maximize that panel: You can also press Ctrl-Backspace to minimize, or Shift-Escape to maximize. When you maximize a panel, it expands to fill the entire screen. When you minimize a panel, it goes away except for a tab on the left side: Page 19

22 Saving and Opening Layouts You can also drag any of the panels to any desired position, and the rest of the workspace will shift to accommodate the changes. Saving your customized layout 1. Choose Layout > User Defined > Save to Server. 2. Enter a name for the new layout. 3. Click OK. The saved window layout includes panel size, location, and genomic view. Opening your customized layout 1. Choose Layout > User Defined > Open from Server. 2. Select the layout to load. 3. Click OK. Note: You must be viewing the same secondary analysis data as when you saved the layout. You can also switch to one of 4 predefined layouts using Layout Menu commands: Minimal Layout: Displays 2 or 3 panels (Genome, Region, Details) horizontally. This layout is based on the data being visualized. Overview Layout: Displays 2 or 3 panels (Genome, Region, Details) vertically on the left, with the Table Browser, User Tab and Selection tab on the right. Details Layout: Displays the Region and Details panels horizontally, with the Details panel taking up most of the screen. Annotate and Share Layout: Displays the Genome panel vertically on the left, with the other panels and tabs shown horizontally on the right. Page 20

23 Menu Commands File Menu View Menu Layout Menu Open Data from Server: Opens one of three types of files for display: Job Metadata (.rdf), Alignment (cmp.h5), or Reference Metadata (reference.info.xml). See Opening Data Files Directly from SMRT View on page 4 for details. Add Tracks: Imports annotation files from your local PC or network, in GFF3, GTF, VCF, or GZ format. The annotation track is added to the existing display. Add Tracks From Server: Imports annotation files from the server where SMRT Analysis is installed, in GFF3, GTF, VCF, or GZ format. The annotation track is added to the existing display. Save Project: Saves your current session as a project (.project.jnlp). This includes references to the data you were looking at, the layout you last used, the genomic coordinates, the font size, and the settings that were in effect at the time. Export Data: Exports all data in the Table Browser, Region panel, or Details panel as a GFF3 file, including data that scrolls offscreen. The exported data includes annotations, tracks, genes, variants, and all reads. Export Graphics: Exports the data in specified panels as separate PDF or PNG graphic files, including data that scrolls off-screen. Printer Settings: Displays the Page Setup dialog where you specify which printer to use, page orientation, paper size, and so on. Print: Displays a dialog where you specify which panel contents to print. All data in the selected panel(s) print, including data that scrolls off-screen. Exit: Exits SMRT View. Autofit Views to Panels: Resets all panels so that the data fill up the panel. This command does not affect the size or location of the individual panels. Reset to Default Genomic Range: Resets the genomic range in all panels to the default range. This command does not affect the size or location of the individual panels. Show Tracks: Displays data tracks. Show Feature Labels: Display labels for genes, annotations, and tracks. Magnifier: Changes the display magnification used in the currently-selected panel. Minimal Layout: Displays 2 or 3 panels (Genome, Region, Details) horizontally. This layout is based on the data being visualized. Overview Layout: Displays 2 or 3 panels (Genome, Region, Details) vertically on the left, with the Table Browser, User Tab and Selection tab on the right. Details Layout: Displays the Region and Details panels horizontally, with the Details panel taking up most of the screen. Page 21

24 Annotate and Share Layout: Displays the Genome panel vertically on the left, with the other panels and tabs shown horizontally on the right. User Defined Open from Server: Displays a list of saved window layouts. When you select a saved layout and click OK, it replaces the current window layout. Save to Server: Displays a dialog where you save the current application layout under a new name. The saved window layout includes panel size, location, and genomic view. Bookmarks Displays the Bookmarks dialog where you can save and open bookmarks. Use bookmarks to save specific regions of interest for later examination. A bookmark includes references to the data you were looking at, the layout you last used, the genomic coordinates, the font size, and the settings that were in effect at the time. Note: Bookmarks can be shared across jobs only if the reference is the same, and jobs have the same number and types of panels, as the bookmark saves the layout. To create a bookmark: Click the Create (push pin) button, then enter a note describing the contents of the bookmark. To save bookmarks so that they persist after you exit SMRT View: Click the Save button. To open a saved bookmarks file: Click the Open button. Tools Menu Table Browser Displays data in table format, using one of three viewing options: All Annotations (Displays all data except the individual reads shown in the Details panel.) Selected Contig (Displays only data from the currently-selected contig.) Details Data (Display only data currently visible in the Details panel.) Go To Location Displays the Go To dialog, where you specify a starting and ending position for the genomic range to view. The range you specify displays in the Region and Details panels. If the visualization includes multiple genomes, chromosomes, or segments, you first select which contig to navigate to. Session Properties Displays the Session Properties dialog, which displays information about the current session, including: The files that are visualized. Page 22

25 Track and reference information. Number of data points. The server and clients used. Install SMRT View Installs on your local computer (Windows/Macintosh/Linux) a desktop shortcut to the version of SMRT View on the remote server where SMRT Analysis is installed. When you double-click the shortcut, SMRT View opens in a limited mode where you can only choose File > Open Data from Server. Tools Menu, Preferences Displays the Preferences dialog where you set application preferences. Changes to many preferences are applied immediately, so you can quickly see how they affect the visualization. To close the dialog and accept the changes: Click OK. The changes will not be in effect the next time you start SMRT View. To save your changes so that they are in effect when you restart SMRT View: Click Save. To reset all preferences to their default values: Click Global, then click Restore Defaults. (Changes take effect after you restart the application.) Variants Annotations - Color This setting affects the colors associated with individual DNA bases used to display normal annotations, substitutions, deletions, and insertions in the Details panel. To change a color: Click it, select a different color, then click OK. Variants Annotations - Filter Base Annotations by Variants When unchecked, base-level annotations are highlighted; that is they are not filtered. When checked, base-level annotations are not highlighted unless they occur at the same genomic location of a variant where the coverage is at least a number that you enter, between 1 and 10. This setting affects the Details panel only, and can be toggled on and off from the toolbar. Variants Annotations - Filter Variants by Coverage When unchecked, variants are not filtered. When checked, variants are filtered based on the number that you enter, between 0 and 100. The higher the number, the higher the coverage must be to view the variant. This setting affects all panels. Page 23

26 QV Annotations When Show Base QV Annotations is unchecked, quality values are not used to change the color of annotations. When Show Base QV Annotations is checked, quality values are used to change the color intensity of annotations based on the numbers that you enter in the Threshold column. Base QV thresholds are on a per-read and per-base basis, and come from primary analysis. Not all files include QV thresholds, so this option may not be applicable in all cases. The colors displayed are derived from the colors you selected for the Variants Annotations. When Show Map QV Annotations is checked, the color of reads change to display the calculated Phred-scale read map quality value. This value estimates the probability that a read is incorrectly mapped to its displayed mapped location. When genomes are repetitive, reads sampled from within repeat regions are likely to have a higher probability of mismapping - this is indicated by a low map quality value. Alternatively, regions annotated as repeats may still be reliably mapped to - this is indicated by a high map quality value. Show Map QV Annotations can be toggled on and off from the toolbar. Note: If you have both options checked: Base QV annotation takes precedence when you are zoomed in. Map QV annotation takes precedence when you are zoomed out. Base Modifications When Show Kinetogram and Base Modification Annotations is checked, bases are annotated with one of three types of annotation: Variants and Base QV, Raw Interpulse Duration (IPD), or Pulse Width. Note: The next 3 options do not apply if you selected Variants and Base QV: Choose the type of scaling to use for the display: Linear, Log10, Square Root, or Exponential. The color display on the right changes based on the scaling you select. Specify the minimum and maximum kinetic measurements to display. The color display on the right changes based on the values you enter. Change the colors of the reverse or forward strand: Double-click the upper part (forward strand) or lower part (reverse strand) of the color bar. When Filter Modification Events by Confidence is checked, base modification events display only if they are at least the number that you enter, between 1 and 100. The higher the number, the higher the coverage must be to view the event. Base modification information displays in the Region panel as additional tracks, as well as in the Details panel. The setting can be toggled on and off from the toolbar. Page 24

27 Single Molecule Annotations When Show Single Molecule Structure Information is checked, the visual display changes to include structure annotation information associated with the reads. Draw Connecting Lines draws lines between connecting subreads. (You may have to wait a bit for the additional information to be retrieved from the server.) When Structures Only is checked, only reads that are part of a structure display. Details Panel - Data Reading Mode Reads Only: Retrieves only reads data from the remote SMRT View server. Does not retrieve base sequences or annotations. Reads+Bases Dynamic: Retrieves only reads data from the remote SMRT View server when you are zoomed out. When you zoom in to the single DNA base level, it automatically updates the reads with bases data after approximately 2 seconds. Reads+Bases Always: Retrieves fully-annotated reads from the remote SMRT View server. Note: This setting retrieves lots of data from the server and can affect SMRT View performance. Details Panel - Genomic Range Limits Fixed: Specifies the maximum genomic range to display in the Details panel when you have zoomed all the way out. The range is from 1,000 bp to 10,000,000 bp. Dynamic: Specifies the maximum number of bases to display in the Details panel when you have zoomed all the way out. The range is from 1,000 to 50,000,000 bases. Note: The larger the range or number of bases that you display, the more memory SMRT View will use and the longer the wait time will be. You may want to use a smaller range or number if SMRT View becomes slow to load files, especially files with high coverage. Details Panel - Max Bases When Genomic Range Limits Fixed is selected, specifies the genomic range to display in the Details Panel. The range is from 1K to 10M bp. When Genomic Range Limits Dynamic is selected, specifies the maximum number of bases to visualize. The genomic range adjusts automatically based on coverage. The range is from 1K to 50M bases. Details Panel - Coverage Limit Specifies limits for maximum coverage in 1K increments; a lower limit increases performance. The default coverage is 1K, with a maximum of 15K. Details Panel - Show Tooltips for Reads When checked, displays tooltips when you move the mouse over the Details panel. Page 25

28 This setting affects the Details panel only. Global Panel - Link the Genome and Region panels When linked, changes to the Genome panel are reflected in the Region panel. When unlinked, information displayed in the Region panel is independent of what you select in the Genome panel. Global Panel - Link the Region and Details panels When linked, changes to the Region panel are reflected in the Details panel. When unlinked, information displayed in the Details panel is independent of what you select in the Region panel. Notes: Changes to these two preferences are reflected on the appropriate toolbars and in the Go To dialog. Linking/unlinking changes that you make on the toolbars are reflected in the preferences. When you select a different contig, the Region and Details panels always display that contig, whether or not the panels are linked. Global Panel - Always show memory usage in status bar When checked, displays memory usage information in the lower right-hand portion of the screen. When unchecked, memory information displays only if you have less than 20% memory available. Help Menu Help: Displays the online help file. PacBio Online: Uses your default web browser to access Pacific Biosciences. PacBio DevNet: Uses your default web browser to access Pacific Biosciences' DevNet developer site. About SMRT View: Displays version information. Page 26

29 Right-Click Commands Genome/Region/Details Panel Zoom In: Displays a smaller area of the currently-selected panel. Zoom Out: Displays a larger area of the currently-selected panel. Zoom to Base: Displays at single-base resolution. (Details panel only.) Previous Genomic Range: Redisplays the panel genomic range that existed before you scrolled, zoomed in or out, and so on. Default Genomic Range: Resets the view to the default genomic range for the selected panel. View Insertions: Displays a dialog showing insertions in a selected region. (Details panel only.) Autofit Views to Panels: Resets all panels so that the data fill up the panel. This command does not affect the size or location of the individual panels. Decrease Height of DNA Letters: Decreases the character size used to display the letters ACGT. (Details panel only.) Increase Height of DNA Letters: Increases the character size used to display the letters ACGT. (Details panel only.) Decrease Height of Kinetogram: Decreases the height of the kinetogram when displaying base modifications. (Details panel only.) Increase Height of Kinetogram: Increases the height of the kinetogram when displaying base modifications. (Details panel only.) Decrease Height of Tracks: Decreases the height of individual tracks, including the Reference track. (Region and Details panel only.) Increase Height of Tracks: Increases the height of individual tracks, including the Reference track. (Region and Details panel only.) Magnifier: Changes the magnification used in the selected panel. Table Browser Panel All Annotations: When checked, displays all data except the individual reads shown in the Details panel. Selected Contig: When checked, displays only data from the currently-selected contig. Details Data: When checked, displays only data currently visible in the Details panel. Fit Columns to Panel: When checked, automatically resizes the table to fit the Table Browser panel. Selection Panel Small Font/Medium Font/Large Font: Specifies the font size to use in the Selection panel. Page 27

30 Keyboard Commands The following keyboard commands work in the currently-selected panel (Genome, Region, or Detail.) Keyboard Command Plus (+) Minus (-) Left Arrow Right Arrow Up Arrow Down Arrow Page Up Page Down Enter Home End F1 F6 t T d D k K Double-click a panel s title bar CTRL-Backspace Shift-Escape Alt-F4 Action Zoom in: Display a smaller area of the currentlyselected panel. Zoom out: Display a larger area of the currentlyselected panel. Scroll left in the genomic range of the currentlyselected panel. Scroll right in the genomic range of the currentlyselected panel. Move to the previous marker in the track. Move to the next marker in the track. Move up a specified percent. Move down a specified percent. Reset the visible range to the default for the selected panel. Go to the start of the genomic range. Go to the end of the genomic range. Display help. Move the focus to the next panel. Decrease the height of tracks. Increase the height of tracks. Increase the character size used to display the letters ACGT. Decrease the character size used to display the letters ACGT. Decrease the height of the kinetogram when displaying base modifications. Increase the height of the kinetogram when displaying base modifications. Expand the panel to fill the screen. Double-click again to contract the panel to its default position. Minimize the selected panel. Maximize the selected panel. Exit SMRT View. Page 28

31 Toolbar Commands Pan Scroll left or right in the genomic range of the currently-selected panel. Zoom Zoom in: Display a smaller area of the currently-selected panel. Zoom out: Display a larger area of the currently-selected panel. Zoom to Base: Display at single-base resolution in the Details panel only. Go To Displays the Go To Location dialog. This is a useful way to navigate to a known genomic location. If the data includes multiple genomes, chromosomes, or segments, select a contig from the Contig list. Go to a specific location in the Genome panel: Enter a starting position and an ending position, then click the >> button. (Optional) Go to a specific location in the Region or Details panel: Enter starting and ending positions, then click the >> button. (Note: These fields are available only if the Region and/or Details panels are unlinked.) Feature Select the previous or next feature in the currently-selected panel. Page 29

32 Map QV Toggles the Show Map QV Annotations preference on and off. Base Mods Toggles the Show Kinetogram and Base Modification Annotations preference on and off. Page 30

33 Additional Panels Selection Panel This panel displays information about items that you select from the Genome, Region, or Details panel. The items can include contigs, SNP tracks, features, genes, bases, and so on. To view the Selection panel: Click the Selection tab. Displays all possible intersections based on what you selected. When a gene is included in the selection, displays a link for additional information from the National Center for Biotechnology Information (NCBI) web site. Note: For such links to display, you must first add the appropriate annotations to SMRT View. (See Adding Annotation Tracks on page 17 for details.) Selected Gene: Selected Feature: Table Browser SMRT View includes a Table Browser, where you can view data in table format. To do so: 1. Choose Tools > Table Browser. 2. Select one of the three viewing options: Page 31

34 All Annotations (Displays all data except the individual reads shown in the Details panel.) Selected Contig (Displays only data from the currently-selected contig.) Details Data (Display only data currently visible in the Details panel.) 3. Click the tab at the left of the screen to display the Table Browser. To display data in the Region and Details panel: Double-click a table row. To sort data: Click a column header to sort the table on that column. To view columns in different order: Click and drag a column header to move the column. To switch to a different table view: Right-click the table and choose a different viewing option. To resize the table so that it fits the Table Browser: Right-click and choose Fit Columns to Panel. To export table data: Choose File > Export Data and select Table Browser Data. To Filter Visualization Data Using the Table Browser, you can also filter the data displayed in the Region and Details panel. Note: You cannot filter summary features, but you can filter the variance and the modification tracks. 1. Enter text, such as a feature ("ACATCTTT") or a modification type ("m6a") into the entry field. Note: The field is case-sensitive. For information on the valid search strings that you can enter, see util/regex/pattern.html. 2. Click the Eye button. Only the data that you specified displays in the Region and Details panel. (This in addition to filtering the table itself.) You see how many item display out of the total number of items, such as Items: 91 (5,234 Total) 3. To redisplay existing data, click the Eye button again. Page 32

35 Motif Summary Panel This panel displays statistics for the motifs included in the data being visualized. (The information is similar to what you see in the SMRT Portal Base Modifications - Motifs report.) Displays only if the data was generated using the RS_Modification_and_Motif_Analysis protocol and motifs are detected. Page 33

36 Troubleshooting SMRT View takes a long time to display data You may want to set the Details Panel Max Bases preference to a smaller number. (The preference specifies the maximum number of bases to display in the Details panel.) The higher the number, the slower SMRT View may be to load files, especially files with high coverage. To change the setting: Choose Tools > Preferences, click Details Panel, select Fixed Genomic Range or Dynamic Genomic Range, enter a smaller number into the field, then click Close. Every time you try to access SMRT View, the application is downloaded from the server Check the Java temporary file setting to ensure that Java files are being cached. This keeps SMRT View in memory so that you only need to download it once. To check the setting (Windows XP): Choose Start > Settings > Control Panel > Java. On the General Tab, click the Settings button. If necessary, select the keep temporary files on my computer option and click OK. SMRT View does not run when you invoke it If there is a problem starting SMRT View, an error message displays with diagnostic information. In addition, SMRT View creates two log files on the server that you can examine for diagnostic information: /opt/smrtanalysis/common/log/smrtview /opt/smrtanalysis/redist/tomcat/logs Page 34