SMRTlink软件使用手册_三代数据做甲基化资源-CSDN文库

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Page 1

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

SMRT

Link User Guide (v5.1.0)

Page 2

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 3

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.

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