Thermo Fisher Scientific SOLiD™ 3 Software Reference guide

Brand: Thermo Fisher Scientific

Model: SOLiD™ 3 Software

Type: Reference guide

Quick Reference Guide

SOLiD™ 3 Software Operation and Data Analysis

Running Experiments and Analyzing Result Data with ICS, SETS, and SAT

The SOLiD™ 3 System comprises multiple complementary analysis software components that complete primary analysis (image

acquistion, signal processing, color calling and quality control) and secondary analysis (alignment to a reference genome, SNP

identification and base calling) of fragment and mate-paired experiments. This document describes the relationship between the

following software tools used to set and control data analysis, including how and when to use each during your workflow:

Experimental data run on the SOLiD™ 3 System can be auto-exported and analyzed remotely using the offline Data Analysis

Software called GlobalSETS. GlobalSETS encompasses SETS and SAT capability for further analysis.

Note: For more information on GlobalSETS, including its installation and configuration, refer to the SOLiD™ 3 SETS Getting Started

Guide (PN 4389302) or the SOLiD™ Analysis Tools (SAT) User Guide (PN 4392959).

SOLiD™ data analysis can be segmented into primary, secondary, and tertiary analysis. Primary analysis includes processes for

data generation, collection and processing. Secondary and tertiary analyses includes application-specific processing and

visualization tools. The graphic below distinguishes the differences between the various analyses.

Figure 1 Various types of SOLiD™ System data analysis

Note: For complex genomes, such as the human genome, secondary analysis should occur on a different cluster from the one that

controls the SOLiD™ 3 System.

This quick reference card covers:

■Begin a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

■Monitor a run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

■Manage analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

■Perform secondary or tertiary analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

■Report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

SOLiD™ 3 System software suite

Software Application Description and purpose

ICS

(SOLiD™ Instrument Control Software)

A Windows application that controls the operation of the SOLiD™ 3 Analyzer and acquires the

experimental data

SETS

(SOLiD™ Experimental Tracking System)

A browser-based integrated application that enables real-time remote monitoring and visualization of

analysis reports and re-analysis capability

SAT

(SOLiD™ Analysis Tools)

A Linux command line tool that processes the images, generates color calls, performs data filtering,

aligns to a reference, generates base calls, performs SNP identification or custom anaylsis

Page 2

SOLiD™ 3 System Software Operation and Data Analysis

Before you begin

Overview

You can perform one of three types of runs on the SOLiD™ 3 System:

• Workflow analysis (WFA)

• Standard sequencing

• Multiplexed sequencing

Software readiness

All runs require setup using the wizard-driven ICS on the SOLiD™ 3 System workstation computer. Before you start a run on the

SOLiD™ 3 Analyzer, open the SETS software and confirm that the current settings for primary and secondary analysis are the

settings to use for the run, or create a new setting. Also while in SETS load a reference genome to use for your analysis. Secondary

analysis (sequence data alignments to a reference sequence) will not occur without a preloaded reference sequence.

To open SETS, type the SETS software URL. If your configuration is set up correctly, the SETS homepage opens and you are

automatically logged in as a “guest” user. As a guest, you can only view analyzed data and reports. Log in to the software as an

administrator (“Admin”) to perform editing and managing functions.

Run types on the SOLiD™ 3 System

Run type Purpose

WFA • Assess various preparations of templated beads to determine potential quality of sequence data

• Evaluate fraction of P2-positive beads

• Use as a tool to determine deposition density for sequencing slides

• Optimize future sequencing run results on the SOLiD™ 3 Analyzer

• Determine optimal titration point

• Determine bead enrichment efficiency

Sequencing Generate sequencing data for fragment or mate-paired libraries

Multiplexed Generate multiplexed sequencing data for fragment libraries

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SOLiD™ 3 System Software Operation and Data Analysis

Begin a run

Workflow

Workflow steps

1Set primary analysis

settings (SETS)

Primary analysis settings control the traffic light ‘signals,’ (thresholds) that you use to determine the quality

of a particular run cycle. You can modify the value for each threshold setting by:

a. From SETS homepage, select Analysis  Primary Analysis Settings.

b. Click New (hyperlink in the title bar)

c. Name your custom setting.

d. Enter a new value for the Low or High percentage value for any of the listed parameters (panel

alignment, HQ beads, bead rentention, color balance, quality value, blur value, blur population,

exposure value, exposure population and exposure time.

Note: For complete descriptions of each parameter, see the SOLiD™ SETS Getting Started Guide

(PN 4389302).

e. Click Save to keep the new setting for that parameter’s traffic light.

IMPORTANT! The default settings are the Applied Biosysytems recommended settings. However,

you can increase or decrease each value for a custom run setting. Click Restore Defaults at any time

to reestablish the original default settings for primary analysis.

2Set secondary analysis

settings (SETS)

Define the secondary analysis pipelines and parameters as part of your setup for secondary analysis.

a. From SETS homepage, select Analysis  Secondary Analysis Settings.

b. Click New (hyperlink in the title bar) or click Edit to change a setting listed.

c. Name your new setting, then select the reference genome to associate with this setting.

d. Click Edit Pipelines.

e. Scroll over a pipeline’s name to see its description, or click View for a summary of its current settings.

f. Tur n On/Off pipelines as necessary.

g. Click Edit to the right of a pipeline name to edit any active pipeline.

IMPORTANT! Some pipelines are dependent upon others. When you select or deselect one, you may

affect the activation state of the others.

h. Enter new value(s) for the pipeline; click Save and Edit Another Pipeline, or Finish to save and quit.

Note: For more information on pipelines and customizing secondary analysis settings, see the

SOLiD™ Analysis Tools (SAT) User Guide (PN 4392959).

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SOLiD™ 3 System Software Operation and Data Analysis

3Set up email

notification (SETS)

Activating Event Notification generates an email when a run completes or an event occurs, depending on

your specifications.

a. From SETS homepage, select Admin  Change Preferences.

b. Enter the email address (or addresses) where you want to receive event notifications.

c. Select the run-specific events to send email about and the default actions to take in response

(including system errors, fatal run errors, informational updates, and completion updates).

IMPORTANT! When you select “My Runs Only” you will receive email notifications for the runs

created only by you. When you select “All Runs” you will receive email notifications for all the runs on

the instrument.

4Set up auto-export in

preferences (SETS)

a. From SETS homepage, select Admin  Change Preferences.

b. Enter the host name or the IP address of the server where you will export your run data to.

c. Check Auto-export after primary analysis.

d. Click Save.

Note: When a run is created, the “Export to Host” and “Auto-export after primary analysis”

preferences are saved to that run. Any additional changes to the Preferences in the Edit User

Preferences will not affect the values of previous runs.

Note: Run export usually fails because of a change in the host IP address or a loss in the RSA key

files. Manually configure the RSA keys if you encounter such a circumstance.

IMPORTANT! RSA keys must be set up between machines so that the host name can be accepted.

For details on how to set up RSA keys, see the SOLiD™ SETS Getting Started Guide (PN 4389302).

5Load reference

sequence

(SETS)

Secondary analysis will not occur without a preloaded reference sequence. If a specific reference sequence

is not listed in the Reference Genome drop-down list, you can load a new reference before starting a run.

Note: You must be logged into SETS as “Admin” to load new reference sequences.

a. From SETS homepage, select Analysis  Load Reference.

b. Click Browse, then select the appropriate fasta file for your reference sequence.

c. Name the selected file.

Note: The reference sequence name that you enter will appear in the Secondary Analysis screen as a

possible reference genome (in the drop-down list) to select for a run.

d. Click Submit.

STOPPING POINT.After uploading the genome, SETS validates the reference. This validation

process may take several minutes during which the genome is not available. If validation fails, the new

genome is not available in the drop-down list (Secondary Analysis screen).

6Create run and specify

analysis settings

(ICS)

a. From ICS main menu, select Create Run.

b. In the Run Wizard, select Sequencing or Multiplexing as your run type.

c. Name your run.

d. Select the run protocol, primer set, number of bases, analysis settings, and mask configuration to

assign to the run, then click Next.

e. Select the number of samples to apply to the mask.

f. Identify the Sample Name with the correct spot, or position, on the slide.

g. Click Finish.

h. Assign the run to one of the flowcells, or save the run record to run later.

i. Click OK.

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SOLiD™ 3 System Software Operation and Data Analysis

7Detect focus range

(ICS)

Before you start any run, detect, or set, the focal range.

IMPORTANT! If automatically detecting the focal range fails for any reason, follow the steps to manually

detect the focal range found in the SOLiD™ 3 System Instrument Operations Guide (PN 4407430).

a. Close and lock the front doors of the SOLiD™ 3 Analyzer.

b. Open the Imager window (Windows  Imaging System).

c. Select Tool s  Detect Focusing Range.

d. Specify the stage template file by entering the name, or browse to find the correct template file.

Note: The stage template file must match the slide in the target flowcell.

e. Select the flowcell using the drop-down menu, then click Go.

f. After the Imager has completed its auto-focus, it prompts you to save the newly-calculated range;

click Ye s to accept.

g. Validate the new focus range by taking images at random locations.

IMPORTANT! You should see images of beads, ensuring that the algorithm was able to focus on the

beads and not other artifacts. If you do not see bead images, or you see out-of-focus bead images,

set the focus range manually.

8Start the run (ICS) Click Start Run.

Monitor a run

Overview

When a run is in progress on the SOLiD™ 3 System, you can monitor it from either SETS (off-instrument) or ICS (on-instrument).

From ICS you can monitor run conditions by checking the details of the Run Log, view cycle scans and heat maps, or adjust a run in

progress using Run Control. From SETS you can determine the quality of a run by checking the traffic signals in Run Metrics and

view the cycle scans, heat maps, or exposure times.

Workflow

Page 6

SOLiD™ 3 System Software Operation and Data Analysis

Workflow steps

1View email

notifications

Check an email account where you set up event notification(s) to be received for any default actions that

may have occurred.

2View cycle scans

(SETS/ICS)

Monitor the quality of a run by viewing its cycle scans. The cycle scan data is presented in a table listing

each cycle seperately. It displays bead color balance bars indicating percentage of Usable Beads vs. Best

Beads vs. Best + Good Beads for an instant visual assessment of the run’s quality.

(SETS) Access the cycle scans view:

a. Select Run Details page  Overall Reports.

b. Click Cycle Scans to see a full report on all the scans for the selected run.

c. Click Plots to display all the plots for the selected scan.

(ICS) Access the cycle scans view by:

a. In the Task pane, select Cycle Scans  Flowcell 1 or 2.

b. Select a scan to view its sample details (shown in panel below the main table).

c. Toggle between different bead views using options on the Action toolbar.

Note: Refer to the SOLiD™ Data Collection v1.0 Help for information on how to interpret cycle scans.

3View heat maps

(SETS/ICS)

Monitor the quality of a run by viewing its heat maps. Heat maps are generated from both the focal map

image data and ligation image data, providing a color display of all the panels in a run. Heat maps include

bead count, bead signal, and image signal.

(SETS) Access the heat map view by:

a. In the Run Details page, select Overall Reports.

b. Select a scan.

c. Click the HM icon to open a heat map for that scan.

(ICS) Access the heat maps view:

a. Click the Heat Map hyperlink (Cycle Scans  Flowcell) to view a heat map after each ligation.

b. Click Heat Map (Run  Flowcell Details) or Focal Heat Map (Cycle Scans  Flowcell).

Note: Refer to the SOLiD™ Data Collection v1.0 Help for information on how to interpret heat maps.

4View run metrics

(SETS)

Monitor the quality of a run by viewing its run metrics. When you view a run in-progress on the SOLiD™ 3

System, you can determine the quality of each cycle by evaluating the traffic signals for each metric. The

Run Metrics page in SETS displays all metrics per sample for the selected ligation cycle.

(SETS) Access the run metrics view:

a. From the Run Details page, select Run Metrics (link), or from the In-Progress/Most Recent Run

page, select Run Metrics (link).

b. Select the primer set, primer, cycle and version to monitor, then click Display to update the traffic

signals. (Green = pass; Yellow = questionable; Red = fail).

Note: If you encounter red traffic signals in the run metrics during a run, immediately abort the run.

c. Click Get Summary to obtain a full report of all metrics associated with a run.

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SOLiD™ 3 System Software Operation and Data Analysis

5View exposure time

(SETS/ICS)

Monitor the quality of a run by viewing its exposure time. In the SOLiD™ 3 System, every panel could

technically have a different exposure value within a channel. This metric tracks the mean exposure time

over all panels and reports the highest of the four channels.

(SETS) Access the exposure times by:

a. From the Run Details page, select Overall Reports.

b. Click Cycle Scans to see the table view of each channel’s exposure time per cycle.

c. Click Exposure Time Report to view exposure times as a full report.

(ICS) Access the cycle scans view by:

a. In the Task pane, select Cycle Scans  Flowcell 1 or 2.

b. Select a scan to view its sample details (shown in panel below the main table).

c. View the column “Effective Exposure Times (ms)” to see exposure information on all four channels.

6View run logs (ICS) Monitor the quality of a run by viewing its run logs. Each flowcell generates a separate run log that records

instrument events such as fluidics activities and slide scans. Run logs provide start and stop times of

recorded events and indicate pauses and errors during a run.

Run logs include:

•errors

• pauses

• run times

• cycle notation

• general information

(ICS) Access run logs by:

a. From the Task pane, select Cycle Scans  Flowcell Details.

b. Click Run Logs (Action toolbar).

7Pause or stop the run

(ICS)

You can change a run in progress on the SOLiD™ 3 System.

(ICS) To stop a run in-progress:

a. In the Task pane, select Flowcell Details.

b. Click Stop Run (Action toolbar) or select Run Control  Stop Run.

(ICS) To pause a run in-progress:

a. In the Task pane, select Flowcell Details.

b. Select Run Control  Set Early Pause Point.

c. Define the exact primer and cycle step where you want to pause the run, then select the module.

d. Click OK.

8Change the run using

Run Control (ICS)

Run Control allows you to change aspects of a run such as stopping a run, pausing a run, and changing a

run’s progress point.

(ICS) To start a run from any point within the protocol:

a. In the Task pane, select Flowcell Details.

b. Select Run Control  Change Run Progress Point.

c. Select the primer, cycle step, and module from the drop-down options, then click OK.

d. Click Resume Run.

Page 8

SOLiD™ 3 System Software Operation and Data Analysis

Manage analysis

Overview

Managing analysis on a SOLiD™ 3 System consists of a range of activities, from potentially canceling a run midstream due to

questionable quality to exporting satisfactory results after a run has completed. Upon run completion you can determine whether

you want to reanalyze the data or generate reports.

Workflow

Workflow steps

1Cancel analysis (SETS) You may only cancel in-progress analyses from SETS. The analysis will display as an active link (Run Details

view) and show that the run state is complete (i.e., no chemistry is currently running).

a. Click Cancel Analysis for the selected run.

b. Select the analysis to cancel, then click Submit.

Note: To cancel more than one analysis at a time, click Check All, then Submit.

2Re-analyze (SETS) Primary re-analysis starts at the Filter Fasta stage, after color calling. The result of a primary re-analysis is a

new set of FASTA files. Primary re-analysis is applied to all samples in a run.

Secondary re-analysis starts at the Moap-unpaired stage. The result of a secondary re-analysis is

application-specific base space sequences, alignment, and tag counts from the color space reads

(.csfasta). Secondary re-analysis allows you to selectively apply secondary analysis settings to individual

samples.

To perform a primary re-analysis:

a. From the Run Details page, go to the Actions box.

b. Click Reanalyze Primary.

c. When a cycle has more than one scan version, click the radio button next to the version you want to

re-analyze.

Page 9

SOLiD™ 3 System Software Operation and Data Analysis

2Re-analyze (SETS)

(continued)

To perform a secondary re-analysis:

a. From the Run Details page, go to the Actions box.

b. Click Reanalyze Secondary.

c. Check the box next to the samples to re-analyze.

d. Select the Analysis Settings to apply during the re-analysis from the drop-down menu, then click Next.

e. To assign a different reference sequence to a library, uncheck the box “Apply [setting name] reference

to libraries”.

3Preview run metrics

(SETS)

The Run Metrics page in SETS displays all metrics per sample for the selected ligation cycle.

Access the run metrics view:

a. From the Run Details page, select Run Metrics (link), or from the In-Progress/Most Recent Run

page  Run Metrics (link).

b. Select the primer set, primer, cycle and version to monitor, then click Display to update the traffic

signals. (Green = pass; Yellow = questionable; Red = fail)

Note: If you encounter red traffic signals in the run metrics during a run, immediately abort the run.

c. Click Get Summary to obtain a full report of all metrics associated with a run.

4Export results (SETS) You can export data to your local computer or GlobalSETS on a standalone computer cluster, depending on

the size of your experiment. If you opt to export results data to the GlobalSETS, data from one or multiple

SOLiD™ 3 Systems can be accomodated.

Note: Automated data export occurs after primary analysis completes.

There are two ways to export result data with SETS:

•Auto-export

• Manual export

To set up auto-export of run data:

a. From SETS homepage, select Admin  Change Preferences.

b. Enter the host name or IP address of the server where you will export your runs to.

c. Check Auto-export after primary analysis, then click Save.

Note: If an error occurs when attempting to export run data, a manual configuration of the RSA keys

usually resolves the problem. See the SOLiD ™ SETS Getting Started Guide (PN 4389302).

To manually instigate export of run data:

a. From the Run Details page, go to the Actions box.

b. Click Set Up Export.

Note: If the Host has not previously been configured, this link will be disabled. Configure the Host.

c. Select one of the export options (all run data, all run data except color calls, only database records),

then click OK.

d. From the Actions box, click Export.

5Re-analyze (SAT) After exporting data to the GlobalSETS cluster, you can use GlobalSETS (Standalone SETS and SAT) to

automate secondary analysis and generate reports.

Page 10

SOLiD™ 3 System Software Operation and Data Analysis

Perform secondary or tertiary analysis

Overview

After run results have been downloaded and data is present on the standalone computer cluster issue the following command line:

• SAT v3.0 (part of GlobalSETS)

• Corona_Lite

• Application analysis pipeline

GlobalSETS provides SOLiD Applications Interface to integrate custom analysis pipelines into SETS v3.0 and SAT v3.0. This

integration streamlines the analysis to provide optimum performance. Start GlobalSETS from SETS or the SAT command line.

Note: For detailed information, refer to the SOLiD™ SETS Getting Started Guide (PN 4389302) or the SOLiD™ Analysis Tools (SAT)

User Guide (PN 4392959).

Workflow

Offline analysis options

The GlobalSETS software package contains pipelines for matching, pairing, and SNP calling to analyze SOLiD sequencing data

from large genomes. You can also use the SOLiD™ Corona_Lite analysis pipeline and other application analysis tools found online

at the SOLiD™ Software Community. For details, download the Corona_Lite User Guide from the SOLiD™ Software Community.

Note: The SOLiD™ Software Community has sample data sets and open-source software tools on www.solidsoftwaretools.com.

Use this resource for whole genome and targeted resequencing, whole transciptome, de novo sequencing, ChIP-Seq and

methylation analysis.

Page 11

SOLiD™ 3 System Software Operation and Data Analysis

Report

Overview

For a comprehensive picture of an aspect of your run, generate one of the following run reports:

• Matching Summary

•Imaging Metrics

• Heat Map

• Cycle Scans

• Exposure Time

•Flowcell Mask

• WFA (only when applicable)

Reports can be viewed both in real-time and in completed run data.

Workflow

SOLiD™ 3 System Reports

Name Description

Matching Summary Report depicting which of your results match to the reference sequence.

Imaging Metrics Report depicting the cycle scan heat map of all four channels for the selected metric.

Heat Map Report depicting colorized result data mapped to the physical layout on the flowcell slide.

Cycle Heat Map Report depicting cycle scan heat map views of the selected bead type.

Cycle Scans Tabular view depicting all scans per cycle of a run, with links to heat maps and plots.

Exposure Time Report depicting the average exposure time of run cycle scans for each of the four channels.

Flowcell Mask Report depicting a representative view of the physical layout of the sample slide.

WFA Report depicting the results of a Workflow Analysis run as they correspond to each titration.

Part Number 4428109 Rev. A 6/2009

International Sales

For our office locations please call the division

headquarters or refer to our Web site at

www.appliedbiosystems.com/about/offices.cfm

Headquarters

850 Lincoln Centre Drive | Foster City, CA 94404 USA

Phone 650.638.5800 | Toll Free 800.345.5224

www.appliedbiosystems.com

SOLiD™ 3 System Software Operation and Data Analysis

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

NOTICE TO PURCHASER: PLEASE REFER TO THE APPLIED BIOSYSTEMS SOLiD™ 3 SYSTEM SETS GETTING STARTED GUIDE (PN 4389302) FOR LIMITED LABEL LICENSE OR

DISCLAIMER INFORMATION.

Thermo Fisher Scientific SOLiD™ 3 Software Reference guide

Brand: Thermo Fisher Scientific

Model: SOLiD™ 3 Software

Type: Reference guide

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