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SMRT link软件使用手册.pdf
Introduction
PacBio® RS II System Users
Contact Information
Using SMRT® Link
Working with Tables
SMRT Link Menu Commands
Sample Setup
Editing or Printing Existing Calculations
Deleting Existing Calculations
Run Design
Creating a New Run Design
Creating a Run Design by Importing a CSV File
Importing a Run Design
Editing or Deleting Run Designs
Run QC
Default Table Fields
Additional Table Fields
Consumables Table
Data Management
What is a Data Set?
What’s in a Data Set?
Creating a Data Set
Starting an Analysis from a Data Set
Copying a Data Set
Deleting a Data Set
Exporting Data Sets
Viewing Data Set Information
Editing Data Set Information
Data Set QC Reports
What is a Project?
What’s in a Project?
Data Sets and Projects
Creating a Project
Editing a Project
Deleting a Project
Viewing/Deleting Sequence, Reference and Barcode Data
Importing Data
SMRT® Analysis
Creating and Starting an Analysis
Starting an Analysis After Viewing Sequence Data
Canceling a Running Analysis
Viewing Analysis Results
Copying and Running an Existing Analysis
Exporting an Analysis
Importing an Analysis
PacBio® Secondary Analysis Applications
Assembly (HGAP 4) Application
Base Modification Detection Application
Base Modification and Motif Analysis Application
CCS Mapping Application
Circular Consensus Sequences (CCS) Application
Convert BAM to FASTX Application
Convert RS to BAM Application
Demultiplex Barcodes Application
Iso-Seq® Iso-Seq 2 Algorithm Application
Iso-Seq® Algorithm Classify Only Application
Iso-Seq® Iso-Seq 2 Algorithm with Mapping Application
Long Amplicon Analysis (LAA 2) Application
Minor Variants Analysis Application [Beta]
Resequencing Application
Site Acceptance Test (SAT) Application
Structural Variant Calling Application
Working with Barcoded Data
Step 1: Specify the Barcode Setup and Sample Names in a Run Design
Step 2: Perform the Sequencing Run
Step 3: (Optional) Run the Demultiplex Barcodes Application
Step 4: Run Applications Using the Demultiplexed Data as Input
Demultiplex Barcodes Application Details
Symmetric Mode
Asymmetric Mode
Mixed Mode
Visualizing Data Using SMRT® View
Installing 64-bit Java 8 on a Windows Operating System
Installing 64-bit Java 8 on macOS
Using the PacBio® Self-Signed SSL Certificate
Sequel® System Output Files
Configuration and User Management
LDAP
SSL
Adding and Deleting SMRT Link Users
Assigning User Roles
Hardware/Software Requirements
Appendix A - Barcoded Primers
Appendix B - Pacific Biosciences Terminology
General Terminology
Read Terminology
Read Length Terminology
Secondary Analysis Terminology
Accuracy Terminology
For Research Use Only. Not for use in diagnostic procedures.
P/N 101-039-100 Version 05 (May 2018)
© Copyright 2017- 2018, 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
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products. Please refer to the applicable Pacific Biosciences Terms and Conditions of Sale and to the applicable license terms at
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Trademarks:
Pacific Biosciences, the Pacific Biosciences logo, PacBio, SMRT, SMRTbell, Iso-Seq and Sequel are trademarks of Pacific
Biosciences. BluePippin and SageELF are trademarks of Sage Science, Inc. NGS-go and NGSengine are trademarks of GenDx.
FEMTO Pulse and Fragment Analyzer are trademarks of Advanced Analytical Technologies. All other trademarks are the sole
property of their respective owners.
Pacific Biosciences of California, Inc.
1305 O’Brien Drive
Menlo Park, CA 94025
www.pacb.com
SMRT® Link User Guide (v5.1.0)
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
PacBio® RS II System Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Contact Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Using SMRT® Link. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4
Working with Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
SMRT Link Menu Commands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Sample Setup. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7
Editing or Printing Existing Calculations . . . . . . . . . . . . . . . . . . . . . . . .9
Deleting Existing Calculations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Run Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Creating a New Run Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .10
Creating a Run Design by Importing a CSV File . . . . . . . . . . . . . . . . .13
Importing a Run Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .13
Editing or Deleting Run Designs . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14
Run QC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
Default Table Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Additional Table Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
Consumables Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Data Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
What is a Data Set? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
What’s in a Data Set? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Creating a Data Set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Starting an Analysis from a Data Set . . . . . . . . . . . . . . . . . . . . . . . . . 22
Copying a Data Set. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Deleting a Data Set. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Exporting Data Sets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
Viewing Data Set Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
Editing Data Set Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
Data Set QC Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
What is a Project? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
What’s in a Project? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25
Data Sets and Projects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Creating a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Editing a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
Deleting a Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
Viewing/Deleting Sequence, Reference and Barcode Data . . . . . . . . 27
Importing Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .28
SMRT® Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
Creating and Starting an Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
Starting an Analysis After Viewing Sequence Data . . . . . . . . . . . . . . .33
Canceling a Running Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
Page 1
Viewing Analysis Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
Copying and Running an Existing Analysis . . . . . . . . . . . . . . . . . . . . .35
Exporting an Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Importing an Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
PacBio® Secondary Analysis Applications . . . . . . . . . . . . . . . . . . . . . .37
Assembly (HGAP 4) Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
Base Modification Detection Application . . . . . . . . . . . . . . . . . . . . . . .45
Base Modification and Motif Analysis Application . . . . . . . . . . . . . . . .48
CCS Mapping Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
Circular Consensus Sequences (CCS) Application . . . . . . . . . . . . . . .56
Convert BAM to FASTX Application . . . . . . . . . . . . . . . . . . . . . . . . . .58
Convert RS to BAM Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58
Demultiplex Barcodes Application . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
Iso-Seq®/Iso-Seq 2 Algorithm Application . . . . . . . . . . . . . . . . . . . . . .63
Iso-Seq® Algorithm Classify Only Application . . . . . . . . . . . . . . . . . . .68
Iso-Seq®/Iso-Seq 2 Algorithm with Mapping Application . . . . . . . . . . 71
Long Amplicon Analysis (LAA 2) Application . . . . . . . . . . . . . . . . . . . 76
Minor Variants Analysis Application [Beta] . . . . . . . . . . . . . . . . . . . . .79
Resequencing Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85
Site Acceptance Test (SAT) Application . . . . . . . . . . . . . . . . . . . . . . .90
Structural Variant Calling Application . . . . . . . . . . . . . . . . . . . . . . . . . 95
Working with Barcoded Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .98
Step 1: Specify the Barcode Setup and Sample Names in a Run
Design. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Step 2: Perform the Sequencing Run . . . . . . . . . . . . . . . . . . . . . . . . .99
Step 3: (Optional) Run the Demultiplex Barcodes Application . . . . . .100
Step 4: Run Applications Using the Demultiplexed Data as Input . . .101
Demultiplex Barcodes Application Details . . . . . . . . . . . . . . . . . . . . .102
Visualizing Data Using SMRT® View . . . . . . . . . . . . . . . . . . . . . . . . . . .105
Installing 64-bit Java 8 on a Windows Operating System. . . . . . . . . 106
Installing 64-bit Java 8 on macOS . . . . . . . . . . . . . . . . . . . . . . . . . . .106
Using the PacBio® Self-Signed SSL Certificate . . . . . . . . . . . . . . . . . .107
Sequel® System Output Files. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .108
Configuration and User Management . . . . . . . . . . . . . . . . . . . . . . . . . .111
LDAP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
SSL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Adding and Deleting SMRT Link Users . . . . . . . . . . . . . . . . . . . . . . 112
Assigning User Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .112
Hardware/Software Requirements. . . . . . . . . . . . . . . . . . . . . . . . . . . . .114
Appendix A - Barcoded Primers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .115
Appendix B - Pacific Biosciences Terminology . . . . . . . . . . . . . . . . . .116
Page 2
Introduction
This document describes how to use Pacific Biosciences’ SMRT Link
software. SMRT Link is the web-based end-to-end workflow manager for
Sequel Systems. It also supports analysis and management of data from
PacBio RS II systems. SMRT Link includes the following modules:
• Sample Setup: Calculate binding and annealing reactions for
preparing DNA samples. (See “Sample Setup” on page 7 for details)
• Run Design: Design sequencing runs and create and/or import
sample sheets. (See “Run Design” on page 10 for details.)
• Run QC: Monitor run progress, status and quality metrics. (See “Run
QC” on page 15 for details.)
• Data Management: Create Projects and Data Sets; generate QC
reports for Data Sets; view, import, or delete sequence, reference,
and barcode files. (See “Data Management” on page 20 for details.)
• SMRT Analysis: Perform secondary analysis on the basecalled data
(such as sequence alignment, variant detection, de novo assembly,
structural variant calling, and RNA analysis) after a run has
completed. (See “SMRT® Analysis” on page 30 for details.)
SMRT Link also includes SMRT View, a genome browser that displays
sequencing data generated by the PacBio RS II and Sequel Systems.
(See “Visualizing Data Using SMRT® View” on page 105 for details.)
This document also describes:
• The data files generated by the Sequel System for each cell that are
transferred to network storage. (See “Sequel® System Output Files”
on page 108 for details.)
• Configuration and user management. (See “Configuration and User
Management” on page 111 for details.)
• SMRT Link client hardware/software requirements. (See “Hardware/
Software Requirements” on page 114 for details.)
Installation of SMRT Link Server software is discussed in the document
SMRT Link Software Installation (v5.1.0).
New features, fixed issues and known issues are listed in the document
SMRT Link Release Notes (v5.1.0).
PacBio® RS II System Users
SMRT Link’s Data Management and SMRT Analysis modules are
compatible with PacBio RS II data; the rest of the SMRT Link modules
work only with Sequel Systems.
Page 3
• PacBio RS II users should use Binding Calculator, RS Remote, and
RS Dashboard software instead of the SMRT Link Sample Setup,
Run Design and Run QC modules.
• To display only the modules for the PacBio RS II: Choose Configure
from the SMRT Link menu, click Instrument, then check the PacBio
RS II Only box. SMRT Link displays only the Data Management and
SMRT Analysis modules.
Contact Information
For additional technical support, contact Pacific Biosciences at
support@pacb.com or 1-877-920-PACB (7222).
Using SMRT® Link
You access SMRT Link using the Chrome web browser.
• SMRT Link is not available on the instrument – it must be accessed
from a remote workstation.
• Depending on how SMRT Link was installed at your site, logging in
with a user name and password may be required.
• SMRT Link needs a Secure Sockets Layer (SSL) Certificate to ensure
a secure connection between the SMRT Link server and your browser
using the HTTPS protocol.
If an SSL Certificate is not installed with SMRT Link, the application will
use the PacBio self-signed SSL Certificate and will use the HTTP
protocol. In this case, each user will need to accept the browser security
warnings described in “Using the PacBio® Self-Signed SSL Certificate”
on page 107.
After accessing SMRT Link, the Home page displays. (Shown is the
interface for the Sequel System.)
Page 4
• Click the menu to navigate to any of the modules, configure for the
PacBio RS II, view version information, or perform administrative
functions (Admins only).
• Click a module name to access that module. Sample Setup, Run
Design, Data Management and SMRT Analysis include links to
create new Calculations, Run Designs, Data Sets, and Analyses.
• Click the PacBio logo at the top left to navigate back to the SMRT
Link Home page from within the application.
• Click ? to view the SMRT Link Online help.
• Click X to log out of SMRT Link.
• Within a module: Click the module name or the module design to
navigate back to the module’s home screen.
Working with Tables
• To sort table columns: Click a column title.
• To search within a table: Enter a unique search string into the Search
field.
• To show or hide table columns: Click the control, then check or
uncheck column name(s) in the dialog box that displays.
Page 5
SMRT Link Menu Commands
• Home: Displays the SMRT Link Home page.
• Sample Setup: Displays the Sample Setup module. (Sequel System
only.)
• Run Design: Displays the Run Design module. (Sequel System
only.)
• Run QC: Displays the Run QC module. (Sequel System only.)
• Data Management: Displays the Data Management module.
• SMRT Analysis: Displays the SMRT Analysis module.
• Show Alarms...
– Displays SMRT Link system-level alarms. To clear alarms, select
and click Clear Alarm or Clear All Alarms.
• Configure
– To display only the modules for the PacBio RS II: Click Instrument,
then check the PacBio RS II Only box. SMRT Link displays only
the Data Management and SMRT Analysis modules.
– Admin users only: Add/delete SMRT Link users and specify their
roles. See “Adding and Deleting SMRT Link Users” on page 112 for
details.
• About
– Displays software version information and available space on the
server SMRT Link is connected to.
– Click Send to send configuration information to Pacific Biosciences
Technical Support for help in troubleshooting failed analyses.
– Admin users only: Update the SMRT Link Chemistry Bundle,
which includes kit and DNA Control Complex names used in the
Sample Setup and Run Design modules.
Page 6
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