SMRT® View Guide

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

P/N 100-088-600-02

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SMRT® View Guide

Table of Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

Pacific Biosciences® Software Overview . . . . . . . . . . . . . . . . . . . . . . . . 2

Hardware/Software Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Accessing SMRT® View from SMRT® Portal . . . . . . . . . . . . . . . . . . . . . . 3

Installing SMRT® View on Your Desktop . . . . . . . . . . . . . . . . . . . . . . . . . 3

Starting SMRT® View From Your Desktop . . . . . . . . . . . . . . . . . . . . . . 3

Opening Data Files Directly from SMRT® View . . . . . . . . . . . . . . . . . . . . 4

Basic Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Navigating Through Your Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Linking/Unlinking the Region and Details panels. . . . . . . . . . . . . . . . . . 5

Navigating Using Numeric Genomic Locations . . . . . . . . . . . . . . . . . . . 7

Viewing Resequencing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Specifying Variant Annotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Specifying Variant Annotation Colors . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Specifying Quality Value Annotations . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Viewing de novo Assembly Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Viewing Base Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Viewing Insertions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Adding Annotation Tracks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Preparing Annotation Tracks to Add to SMRT® View . . . . . . . . . . . . . 14

Adding Annotation Tracks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Exporting Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Window Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Saving and Opening Layouts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Menu Commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

File Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

View Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Tools Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Tools Menu, Preferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Layout Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Help Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Right-Click Commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Keyboard Commands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Additional Panels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Selection Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Table Browser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

User Data Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Page 1

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 http://www.smrtcommunity.com/SMRT-Analysis/Software/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.

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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.)

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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.

Basic Workflow

SMRT View displays secondary analysis data using three different panels: The Genome panel, Region panel, and the Details panel.

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Navigating Through Your Data

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.

Linking/Unlinking the Region and Details panels

• 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.

• 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.

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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.

The Details panel:

• Allows you to drill down to base-level resolution and visualize SNPs.

• 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. (Zoom in using the + (Plus) key to see the individual bases.) Note: The colors used for the refer-ence sequence bases are different from that of any annotations.

• Displays the direction (forward or reverse) of read strands.• Displays base modifications. See “Viewing Base Modifications”

on page 11 for further details.• Displays insertions. (Select a region, then right-click and choose

View Insertions.)

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• 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 9.

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.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:

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These fields are available only if the Region and/or Details panels are unlinked.)

In any of the 3 panels:

• 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.

Viewing Resequencing Data

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 differ-ences between the reference track and the reads for both strands.

Specifying Variant Annotation

1. Choose Tools > Preferences.2. Click Variants Annotations.3. Check Filter Variant Events 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 pan-els.)

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 Close. The changes take effect immediately.

Specifying Variant Annotation Colors

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 Close. The change takes effect immediately

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 per-

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base 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 Close. The changes take effect immediately. Notes:

• 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.

Viewing de novo Assembly Data

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 vari-ations track (assembled_summary.gff.gz).

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You can also view information about the individual contigs included in the de novo assembly 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.

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• To sort data: Click a column header to sort the table on that column.

Viewing Base Modifications

Base modification data can be helpful in visualizing DNA modification, such as methylation.

Note: The full base modification visualization is possible only if you set up the secondary analysis job in SMRT Portal using:

• Pacific Biosciences' RS_Modification_Detection protocol or• Your own protocol which includes the base modification module.

Base modification mode includes several new visualization tracks:

1. The Modification Regions and Modification Events tracks are shown in the Region panel, both colored by strand.

• The Modifications Regions track displays putative modification events as a bar chart, for both the plus and minus strands. This indicates the number of modification “events”.

• The Modification Events track displays each "event" detected by the analysis as a color-coded marker. (Only events with a confi-dence 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.)

2. IPD Ratio (or normalized IPD) bar charts display in the Details panel, for each position for each strand, next to the reference sequence view with both strands indicated.

• 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.

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• 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 mod-ified. 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.

3. Each aligned read is also shown, separated by strand, in template space. These reads are shown in gray by default to emphasize that they are showing a raw IPD value rather than an IPD ratio. Aligned reads at the bottom of the Details panel are colored by one of two types of annotation: Raw Interpulse Duration or Pulse Width.

4. The reference sequence in the Details panel is split by strand, and a normalized IPD is shown for every position in the alignment, dis-played as colored bars. Pulse width is never shown in the bar chart.

Viewing Base Modifications

1. Choose Tools > Preferences, then click Base Modifications.2. Check Show Base Modifications.3. Select one of the available types of base annotations: Raw Inter-

pulse Duration (IPD) or Pulse Width. (You may have to wait a bit for the additional information to be retrieved from the server.)

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. (Note: Exponential dis-play more strongly emphasizes extreme events.)

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. Your color selection is saved as a prefer-ence for future visualizations.

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.)

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To see all base modification events: Uncheck Filter Modification Events by Confidence.

8. Click Close. The change takes effect immediately.

Note: If you check Show Base Modifications, no other types of annotations display.

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.

• If the Show Base Modification 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.

Adding Annotation Tracks

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.)

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• 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 format.

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.

• 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 pan-

els, in a new track named using the name of the file. The annota-tions do not persist after you close SMRT View.

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Exporting Data

Exporting Graphics

When you export graphics, all data in all panels are exported as individual PNG or PDF image files. This includes data that scrolls off-screen.

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.

Exporting Data You can export data from the 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 Details panel data that scrolls off-screen, as well as annotations, tracks, genes, variants, and all reads.

1. Choose File > Export Data.2. Name the file and choose a location.3. Click Save.

Window Management

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:

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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 Layouts > Save User Layout.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 Layouts > Open User Layout.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 icons on the toolbar:

• Default 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 horizon-tally, with the Details panel taking up most of the screen.

• Annotate and Share Layout: Displays the Genome panel verti-cally on the left, with the other panels and tabs shown horizontally on the right.

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Menu Commands

File Menu • Open Data from Server: Opens one of three types of files for dis-play: Job Metadata (.rdf), Alignment (cmp.h5), or Reference Meta-data (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 net-work, 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 for-mat. 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 Details panel as a GFF3 file, including data that scrolls off-screen. 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 spec-ify 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.

View Menu • 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 cur-

rently-selected panel.

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.)

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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.

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.

• 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.

Session Properties

Displays the Session Properties dialog, which displays information about the current session, including:

• The files that are visualized.• 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.

Note: This command is not available if you are using SMRT Analysis Cloud Beta.

Tools Menu, Preferences

Displays the Preferences dialog where you set application preferences.

• The preferences are applied immediately, so you can quickly see how they affect the visualization.

• After you click Save, any changes you made are saved, and will be in effect the next time you use SMRT View.

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• 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 and then select a different color.

Variants Annotations - Filter Variant Events 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.

Variants Annotations - Filter Base Annotations by Variant Event

• 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.

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 thresh-olds 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 incor-rectly 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.

These settings affects the Details panel only. Show Base QV Annotations can be toggled on and off from the toolbar.

Note: If you have both options checked:

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• Base QV annotation takes precedence when you are zoomed in. • Map QV annotation takes precedence when you are zoomed out.

Base Modifications

• When Show Base Modifications is checked, bases are annotated with one of two types of annotation: Raw Interpulse Duration (IPD) and Pulse Width.

• 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 dis-play. 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. Your color selection is saved as a preference for future visualizations.

• When Filter Modification Events by Confidence is checked, base modification events display only if they are at least the num-ber 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.

• If you check Show Base Modifications, no other types of annota-tions display. The setting can be toggled on and off from the tool-bar.

CCS Annotations

• When Show CCS Structure Information is checked, the visual display changes to include structure annotation information associ-ated with the reads.

• Draw Connecting Lines draws lines between connecting sub-reads. Draw Connecting Lines and Boxes draws additional bounding boxes around the 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.

• Concordant links are drawn in black, while discordant links are drawn in violet.

Details Panel - Data Size Limits

• Fixed Genomic Range: 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 Genomic Range: 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.

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• 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 cover-age.

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.

• You can toggle between Reads+Bases Dynamic and Reads+Bases Always from the toolbar.

Details Panel - Show Tooltips for Reads

• When checked, displays tooltips when you move the mouse over the Details panel.

• 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.

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Layout Menu Predefined Layouts

• Default 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 horizon-tally, with the Details panel taking up most of the screen.

• Annotate and Share Layout: Displays the Genome panel verti-cally on the left, with the other panels and tabs shown horizontally on the right.

User Layouts

• Open User Layout: Displays a list of saved window layouts. When you select a saved layout and click OK, it replaces the current win-dow layout.

• Save User Layout: 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.

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.

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Right-Click Commands

Genome/Region/Details Panel

• Zoom In: Displays a smaller area of the selected panel.• Zoom Out: Displays a larger area of the 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 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.)

• 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.)

• 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 cur-rently-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.

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Keyboard Commands

The following keyboard commands work in the currently-selected panel (Genome, Region, or Detail.)

Keyboard Command Action

Plus (+) Zoom in - display a smaller area of the selected panel.

Minus (-) Zoom out - display a larger area of the selected view.

Left Arrow Scroll left in the genomic range.

Right Arrow Scroll right in the genomic range.

Up Arrow Move to the previous marker in the track.

Down Arrow Move to the next marker in the track.

Page Up Move up a specified percent.

Page Down Move down a specified percent.

Enter Reset the visible range to the default for the selected panel.

Home Go to the start of the genomic range.

End Go to the end of the genomic range.

F1 Display help.

F6 Move the focus to the next panel.

t Decrease the height of tracks.

T Increase the height of tracks.

d Increase the character size used to display the letters ACGT.

D Decrease the character size used to display the letters ACGT.

Double-click a panel’s title bar

Expand the panel to fill the screen. Double-click again to contract the panel to its default position.

CTRL-Backspace Minimize the selected panel.

Shift-Escape Maximize the selected panel.

Alt-F4 Exit SMRT View.

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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 addi-

tional 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 13 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:

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– 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 filter data: Enter text into the Filter field. You see how many item display out of the total number of items, such as Items: 91 (5,234 Total)

• To resize the table so that it fits the Table Browser: Right-click and choose Fit Columns to Panel.

User Data Panel This panel displays links to specific regions of interest within the file.

• To view the User Data panel: Click the User Data tab.• Displays only if you first add the appropriate annotations to SMRT

View. (See “Adding Annotation Tracks” on page 13 for details.)

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Troubleshooting

SMRT View takes a long time to display data

• You may want to set the Details Panel Data Size Limits prefer-ence to a smaller number. (The preference specifies the maximum genomic range or 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 but-ton. If necessary, select the keep temporary files on my com-puter option and click OK.

SMRT View does not run when you invoke it

• If there is a problem starting SMRT View, an error message dis-plays 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

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