Roche AVENIO ctDNA Analysis SW User manual

Roche
Brand
Roche
Model
AVENIO ctDNA Analysis SW
Category
Software
Type
User manual
AVENIO ctDNA Analysis Software
User
Guide
Version 1.0.0
For Research Use Only. Not for use in diagnostic procedures.
Publication information
AVENIO ctDNA Analysis Software User Guide, v1.0.0 2
Publication information
Document material number: 08164231001
Publication version
Software version
Revision date
Change description
1.0.0
1.0.0
March 2017
First version
Edition notice
This publication is intended for users of AVENIO ctDNA Analysis Software.
Every effort has been made to ensure that all the information contained in this publication is correct at the time of publishing. However, the
manufacturer of this product may need to update the publication information as output of product surveillance activities, leading to a new
version of this publication.
Where to find information
This AVENIO ctDNA Analysis Software User Guide contains information about using the analysis software, organized according to the normal
operation workflow.
The AVENIO ctDNA Analysis Software Admin Guide contains information about setting up and managing the analysis software for users.
The AVENIO ctDNA Analysis Kits Reagent Workflow User Guide contains information about using the reagents, organized according to the
normal operation workflow.
General attention
To avoid incorrect results, ensure that you are familiar with the instructions and safety information.
• Pay particular attention to all safety notices.
• Always follow the instructions in this publication.
• Do not use the software in a way that is not described in this publication.
• Store all publications in a safe and easily retrievable place.
Training
Do not carry out operation tasks or maintenance actions unless you have received training from Roche. Leave tasks that are not described in
the user documentation to trained Roche Service representatives.
Screenshots
The screenshots in this publication have been added exclusively for illustration purposes. Configurable and variable data, such as tests, results,
or path names visible therein must not be used for laboratory purposes.
Warranty
Any customer modification to the system renders the warranty or service agreement null and void.
For conditions of warranty, contact your local sales representative, or refer to your warranty contract partner.
Always leave software updates to a Roche Service representative, or perform such updates with their assistance.
Copyright
© 2017 Roche Molecular Systems, Inc. All Rights Reserved.
License information
AVENIO ctDNA Analysis Software is protected by contract law, copyright law, and international treaties. AVENIO ctDNA Analysis Software
contains a user license between Roche Sequencing Solutions, Inc. and a license holder, and only authorized users may access the software and
use it. Unauthorized use and distribution may result in civil and criminal penalties.
Publication information
AVENIO ctDNA Analysis Software User Guide, v1.0.0 3
Open source and commercial software
The AVENIO ctDNA Analysis Software may include components or modules of commercial or open-source software. For further information on
the intellectual property and other warnings, as well as licenses pertaining to the software programs included in the AVENIO ctDNA Analysis
Software, refer to the OTSS Annex AB electronic distribution included with this product.
This open source and commercial software and AVENIO ctDNA Analysis Software as a whole can constitute a device regulated in accordance
with applicable law. For more detailed information, refer to the user manual and labeling.
Please note that the respective authorization is no longer valid according to the corresponding legislation should any unauthorized changes be
made to AVENIO ctDNA Analysis Software.
Trademarks
The following trademarks are acknowledged:
AVENIO is a trademark of Roche.
All other trademarks are the property of their respective owners.
Patent license limitations
For patent license limitations, go to www.technical-support.roche.com and search by product name or product material number. License
disclaimers are listed at the bottom of the Basics tab.
Feedback
Every effort has been made to ensure that this publication fulfills the intended use. All feedback on any aspect of this publication is welcome
and is considered during updates. Contact your Roche representative, should you have any such feedback.
Contact addresses
Manufacturer
Roche Molecular Systems, Inc.
1080 US Highway 202 South
Branchburg, NJ 08876
USA
Distribution
Roche Diagnostics GmbH
Sandhofer Strasse 116
68305 Mannheim
Germany
Distribution in USA
Roche Diagnostics
9115 Hague Road
Indianapolis, Indiana 46256
USA
Technical support
For product-related technical documents or to contact Roche Technical Support, go to sequencing.roche.com/support.
Publication information
AVENIO ctDNA Analysis Software User Guide, v1.0.0 4
Table of contents
Publication information ....................................................................................................................................... 2
Contact addresses ................................................................................................................................................................................................ 3
Technical support .................................................................................................................................................................................................. 3
Table of contents ................................................................................................................................................................................................... 4
Preface ...................................................................................................................................................................................................................... 6
Regulatory disclaimer ..................................................................................................................................................................................... 6
Symbols and abbreviations........................................................................................................................................................................... 6
Chapter 1. Getting started ................................................................................................................................. 7
About AVENIO ctDNA Analysis Software ..................................................................................................................................................... 7
Logging on to the analysis software ............................................................................................................................................................... 7
To log on ............................................................................................................................................................................................................. 7
Logging off of the analysis software ............................................................................................................................................................... 9
To log off ............................................................................................................................................................................................................. 9
Changing your password .................................................................................................................................................................................... 9
To change your password............................................................................................................................................................................. 9
Resetting your password .................................................................................................................................................................................. 10
To reset your password ............................................................................................................................................................................... 10
Changing your display name ........................................................................................................................................................................... 10
To change your display name.................................................................................................................................................................... 10
Updating your email address .......................................................................................................................................................................... 11
To update your email address ................................................................................................................................................................... 11
Chapter 2. Overview.......................................................................................................................................... 12
Analysis software process ................................................................................................................................................................................ 12
Analysis software user interface .................................................................................................................................................................... 12
Dashboard ........................................................................................................................................................................................................ 12
Sample Entry ................................................................................................................................................................................................... 13
Run Settings .................................................................................................................................................................................................... 14
Analysis ............................................................................................................................................................................................................. 14
Results ............................................................................................................................................................................................................... 15
Chapter 3. Create runs ..................................................................................................................................... 16
Creating a run manually .................................................................................................................................................................................... 16
To create a run manually ............................................................................................................................................................................ 16
Creating a run using the CSV template ....................................................................................................................................................... 18
To download the CSV template ................................................................................................................................................................ 18
To upload the CSV file for your run ......................................................................................................................................................... 18
Choosing the analysis input file and starting analysis ............................................................................................................................ 19
To choose a BCL analysis input file ......................................................................................................................................................... 19
To choose FASTQ analysis input file ....................................................................................................................................................... 21
Editing sample information .............................................................................................................................................................................. 22
To edit sample information ......................................................................................................................................................................... 22
Removing a sample from a run ...................................................................................................................................................................... 23
Setting the output directory for Analysis files ............................................................................................................................................ 23
To set the output directory for Analysis files ........................................................................................................................................ 23
Chapter 4. Manage runs .................................................................................................................................. 24
Viewing runs and run status ............................................................................................................................................................................ 24
To view recent runs and their run status ............................................................................................................................................... 25
To view all runs and their run status ....................................................................................................................................................... 25
To search for runs ......................................................................................................................................................................................... 26
Deleting a run ....................................................................................................................................................................................................... 26
To delete a run................................................................................................................................................................................................ 26
Cancelling a queued or in-progress run ..................................................................................................................................................... 26
To cancel a queued or in-progress run.................................................................................................................................................. 26
Duplicating and rerunning an analysis ........................................................................................................................................................ 27
To duplicate and rerun an analysis.......................................................................................................................................................... 27
Viewing run results ............................................................................................................................................................................................. 27
To view run results ........................................................................................................................................................................................ 27
Chapter 5. View sample results ...................................................................................................................... 29
Viewing individual sample results ................................................................................................................................................................. 29
To view individual sample results ............................................................................................................................................................. 29
Publication information
AVENIO ctDNA Analysis Software User Guide, v1.0.0 5
Viewing SNV and Indel results ....................................................................................................................................................................... 30
To view SNV and Indel results................................................................................................................................................................... 30
Viewing individual variant information ......................................................................................................................................................... 32
To view individual variant information .................................................................................................................................................... 32
Viewing CNV and Fusion results .................................................................................................................................................................... 33
To view CNV and Fusion results ............................................................................................................................................................... 33
Viewing Loci of Interest..................................................................................................................................................................................... 34
To view the Loci of Interest ........................................................................................................................................................................ 34
Chapter 6. Manage reports ............................................................................................................................. 36
Viewing an automatically generated report ............................................................................................................................................... 36
To view an auto-generate report .............................................................................................................................................................. 36
Generating custom reports .............................................................................................................................................................................. 37
To generate a custom report by using a saved filter set .................................................................................................................. 37
To generate a custom report by choosing your filter settings ........................................................................................................ 39
Interpreting the variant report ......................................................................................................................................................................... 40
Sample summary ........................................................................................................................................................................................... 40
Results summary ............................................................................................................................................................................................ 41
Run details ....................................................................................................................................................................................................... 41
Variant details ................................................................................................................................................................................................. 41
Viewing sample metrics .................................................................................................................................................................................... 42
To view sample metrics ............................................................................................................................................................................... 42
Sample Metrics report interpretation ...................................................................................................................................................... 44
Run metrics ...................................................................................................................................................................................................... 44
Chapter 7. View output files and run metadata .......................................................................................... 45
Viewing output files ............................................................................................................................................................................................ 45
To view output files ....................................................................................................................................................................................... 45
Viewing run metadata ........................................................................................................................................................................................ 46
To view run metadata ................................................................................................................................................................................... 46
Viewing sample metadata ................................................................................................................................................................................ 47
To view sample metadata ........................................................................................................................................................................... 47
Appendix A. Troubleshooting .......................................................................................................................... 48
User input errors .................................................................................................................................................................................................. 48
System errors ........................................................................................................................................................................................................ 49
To troubleshoot system errors ................................................................................................................................................................... 49
Workflow errors ................................................................................................................................................................................................... 50
To troubleshoot workflow errors .............................................................................................................................................................. 50
Error information ............................................................................................................................................................................................ 51
Downloading error logs ..................................................................................................................................................................................... 51
Appendix B. Methodology ............................................................................................................................... 52
Appendix C. Converting a BCL output file to FASTQ .................................................................................. 53
To convert a BCL file to FASTQ ................................................................................................................................................................. 53
Publication information
AVENIO ctDNA Analysis Software User Guide, v1.0.0 6
Preface
Regulatory disclaimer
For Research Use Only. Not for use in diagnostic procedures.
AVENIO ctDNA Analysis Kits
The AVENIO ctDNA Analysis Kits are a portfolio of three next-generation sequencing liquid biopsy assays designed to
help researchers understand the genomic complexity of aberrations in solid tumors. The kits are for research use only,
not for use in diagnostic procedures. The AVENIO ctDNA Analysis Kits provide an end-to-end tumor profiling and
monitoring solution, from cfDNA extraction to analysis and variant reporting.
AVENIO ctDNA Analysis Software
The AVENIO ctDNA Analysis Software workflow provides a complete set of tools required for data analysis and variant
reporting when used with the AVENIO ctDNA Analysis Kits. This includes primary analysis with customized
workflows, intuitive variant calling, filtering, and reporting of results. This post-sequencing analysis software makes it
easy for labs to analyze samples and quickly access the genomic information they require.
Symbols and abbreviations
AVENIO ctDNA Analysis Software
Except where the context clearly indicated otherwise, the following product names and descriptors are used.
Product name
Descriptor
AVENIO ctDNA Analysis Software
analysis software
Symbols used in the publication
Explanation
Safety alert symbol: The safety alert symbol is used to alert you to potential physical injury hazards. To avoid possible injury
or death, comply with all safety messages that follow this symbol.
Important note: Highlights information that is critical for optimal performance of the system. May also indicate that loss of
data or invalid data could occur if the precautions or instructions are not observed.
Information note: Identifies items of general interest and additional information about the topic or procedure being
described.
Abbreviations
The following abbreviations are used.
Abbreviation
Definition
AF
Allele Frequency or Allele Fraction
BCL
Base Call File
cfDNA
cell free DNA
ctDNA
circulating tumor DNA
CNV
Copy Number Variant
GID
Group ID
Indel
Insertion or deletion
SNV
Single Nucleotide Variation
UID
User ID
Chapter 1. Getting started
AVENIO ctDNA Analysis Software User Guide, v1.0.0 7
Chapter 1. Getting started
This guide includes information and instructions for using the AVENIO ctDNA Analysis Software to create and
manage runs, view sample results, manage reports, and view output files and run metadata. This guide also includes
general troubleshooting information and methodology.
About AVENIO ctDNA Analysis Software
AVENIO ctDNA Analysis Software enables the end-to-end ctDNA analysis kit workflow. This solution includes sample
preparation, sequencing, analysis run setup, bioinformatics workflows, and variant reporting. The AVENIO ctDNA
Analysis Software allows labs to quickly and easily transform enriched circulating tumor DNA samples processed on an
Illumina NextSeq into meaningful insights for profiling tumor mutations and monitoring how they evolve over time.
The ctDNA Analysis Software is run on a server that is connected to on-premise shared network storage. The Oncology
Analysis Server is required and also available from Roche. After the AVENIO ctDNA Analysis Software completes the
workflow and report, the shared network storage will contain both the sequencing output files and the analysis output
files. The figure below illustrates the software and hardware necessary to run the AVENIO ctDNA Analysis Software. It
highlights the bioinformatics workflow, user interface, and PDF reports available at the end of a successful analysis run.
Logging on to the analysis software
Prerequisites:
Your admin set up user accounts for your system.
Google Chrome version 51.0 or later or Mozilla Firefox version 46.0 or later browser is installed.
Your admin provided you with the analysis software URL.
To log on
1. Open your Chrome or Firefox browser, and navigate to the analysis software URL provided to you by
your admin.
Chapter 1. Getting started
AVENIO ctDNA Analysis Software User Guide, v1.0.0 8
The first time you open the URL, you will get a privacy message similar to the one below but with a different
IP address.
2. Choose Advanced.
3. Choose the link Proceed to <your IP address> (unsafe).
The analysis software opens to the Log On page.
4. Enter your credentials. (If you forgot your password, see Resetting your password.)
5. Accept the Terms and Conditions.
If you reset your browser cache or use a new browser, you will need to accept the
Terms and Conditions again
before logging on to the system.
Chapter 1. Getting started
AVENIO ctDNA Analysis Software User Guide, v1.0.0 9
6. Choose Log On.
The analysis software opens to the Dashboard.
Users are automatically logged out after 24 hours.
Logging off of the analysis software
To log off
1. At the upper right corner, choose your name.
2. Choose Log Off.
Changing your password
To change your password
1. Log on to the analysis software.
2. At the upper right corner, choose your name, and choose My Account.
3. Change your password, as desired.
4. Choose Change Password.
Chapter 1. Getting started
AVENIO ctDNA Analysis Software User Guide, v1.0.0 10
Resetting your password
If you forgot your password, you can reset it on the analysis software Log On page.
To reset your password
1. On the analysis software Log On page, choose Forgot Password.
2. Enter your email address, and choose Reset.
You will receive an email with a link to reset your password. Follow the instructions in the email.
Changing your display name
You can change the way your name appears to others in the analysis software.
To change your display name
1. Log on to the analysis software.
2. At the upper right corner, choose your name, and choose My Account.
3. Change your Real name, as desired.
4. Choose Save.
Chapter 1. Getting started
AVENIO ctDNA Analysis Software User Guide, v1.0.0 11
Updating your email address
The analysis software uses email for logging on to the software, resetting passwords, and sending automatic
run-complete notifications.
To update your email address
1. Log on to the analysis software.
2. At the upper right corner, choose your name, and choose My Account.
3. Update your email address, as needed.
4. Choose Save.
Chapter 2. Overview
AVENIO ctDNA Analysis Software User Guide, v1.0.0 12
Chapter 2. Overview
Analysis software process
The analysis software assumes that you started the sequencer, completed sequencing, and are ready to set up
bioinformatics analysis.
The following is a high-level overview of the analysis software process:
Create an analysis run using sample information from your sequencing run (see Creating a run manually or
Creating a run using the CSV template).
Choose the sequencing output files and start analysis (see Choosing the analysis input file and starting analysis).
View runs and run status, duplicate and rerun an analysis, view run results, and more (see Manage runs).
View the results in the user interface (see View sample results) or view the generated reports (see Viewing an
automatically generated report).
Analysis software user interface
The analysis software includes the following main structure:
Dashboard
Sample Entry page
Run Settings page
Analysis page
Results page
Dashboard
From the Dashboard, you can:
• Create a new run
• View runs
Each run displays a run tile that includes a color indicator that represents the state of the run. (For a detailed description
of color indicators and run states, see Viewing runs and run status.) If you have not yet created runs, the dashboard
displays just the headers for each status: Setup, Running, and Completed.
You can go to the Dashboard from anywhere in the software by choosing the icon located at the top left
of any page.
Chapter 2. Overview
AVENIO ctDNA Analysis Software User Guide, v1.0.0 13
Sample Entry
The first step for creating a new run is to enter sample information. Use the Sample Entry page to enter the run name
and sample information for the run.
Enter samples manually—one at a time—or download the provided CSV template, enter your sample information,
and upload the file.
The choice of panel type from the Panel drop-down list determines which analysis workflows are available for
the sample.
The samples you entered are added to the sample table at the bottom of the Sample Entry page.
The information in the sample table is shared between the Manual Entry tab and the CSV Upload tab, so that all of your
sample information is included in one table.
Once a Sample Adapter is used for a particular run, it is no longer available from the Sample Adapter drop-down list.
Chapter 2. Overview
AVENIO ctDNA Analysis Software User Guide, v1.0.0 14
Run Settings
On the Run Settings page, select the sequencing files you want to associate with the samples you entered for your run,
the output directory for your analysis files, and the email addresses of users that want run-complete notifications.
Analysis
The Analysis page displays the status of the run, which includes the amount of time that has elapsed since analysis
started, and the status of each sample. This page displays only the running or queued run.
Chapter 2. Overview
AVENIO ctDNA Analysis Software User Guide, v1.0.0 15
Results
The Results page displays high-level analysis results for each sample including the total number of unfiltered
variants reported for the sample, and a subset count of variants that passed the filters as set by the analysis workflow
for the sample.
View basic metadata about the run by choosing the icon.
Chapter 3. Create runs
AVENIO ctDNA Analysis Software User Guide, v1.0.0 16
Chapter 3. Create runs
Create a new run manually—by adding one sample at a time—or use the provided CSV template to upload multiple
samples at once.
A run created in the analysis software represents a sequencing run.
I
ndividual samples cannot be analyzed one at a time. The run must be analyzed with all
samples specified.
Creating a run manually
To create a run manually
1. From the dashboard, choose Create New Run.
2. In the Run Name field, enter the run name using any naming structures assigned by your lab.
3. In the Sample ID field, enter the sample’s ID as assigned by your lab.
Do not use a period or the sequence of letters “sam” or “bam” anywhere in the
Sample ID
.
4. From the Sample Adapter drop-down list, choose the adapter number for the adapter ligated to the sample.
5. In the Isolated DNA Mass field, enter the sample’s isolated DNA mass. If the report metric No. of Mutant Molecules
per mL is desired, in the Plasma Volume field, enter the plasma volume. If this value is not known, select the
Unknown check box.
If you enter more than two decimal places in the
Isolated DNA Mass
and
Plasma Volume
fields, your entries will be rounded up.
The Isolated DNA Mass is the total yield of DNA isolated from the input plasma using the AVENIO cfDNA
Isolation Kit. To determine Isolated DNA Mass, use the following formula, assuming 65µL of DNA Elution Buffer
is applied onto the Filter Tube as specified in the protocol. The concentration used is the Qubit value from the first
QC step, measured after isolating the cfDNA from the plasma.
Isolated DNA Mass (ng) = Concentration of isolated cfDNA (ng/µL) x 65µL
Isolated DNA Mass and
Plasma Volume
are required to calculate No. of Mutant
Molecules per mL of plasma. If the
Isolated DNA Mass and Plasma Volume are not
entered during sample entry on the
Sample Entry page, the overall analysis will be
completed, but this met
ric will not be computed. Isolated DNA Mass is used only for
the calculation of this metric, which depends on the total plasma volume input into
isolation and the total cfDNA mass isolated
. Isolated DNA Mass may not be the
same value as the mass of DNA input into the
library preparation protocol. The
DNA input into the library preparation does not necessarily reflect the starting
material from the plasma, and therefore is not used to calculate No.
of Mutant
Molecules per mL of plasma. Alternatively,
if this metric is not required for the
end
-user, and the user does not know or desire to calculate the Isolated DNA Mass,
any value may be entered as a placeholder, and it will not affect the calculation of
other report metrics.
6. From the Panel drop-down list, choose a panel.
The analysis software includes three panels. The panel you choose determines the available workflow choices in the
Analysis Workflow drop-down list.
7. From the Analysis Workflow drop-down list, choose a workflow. The software comes with 3 Analysis Workflows
(one for each panel). Only admin users can create custom Analysis Workflows.
Chapter 3. Create runs
AVENIO ctDNA Analysis Software User Guide, v1.0.0 17
8. Choose Add.
The sample appears in the sample table at the bottom of the Sample Entry page. It can take up to 5 seconds to add
the sample.
If you need to edit sample information, see Editing sample information
for instructions.
9. Once you have added all samples, choose Continue at the bottom of the page.
The Run Settings page appears. This page can take several seconds to appear.
The
Date Started
,
Machine Name
, and
Flowcell ID
fields appear only if the default
input mount is set in the admin settings.
10. Go to Choosing the analysis input file and starting analysis.
Chapter 3. Create runs
AVENIO ctDNA Analysis Software User Guide, v1.0.0 18
Creating a run using the CSV template
Use the provided CSV template to create a consolidated file of up to 16 samples for your run, then upload the file into
the analysis software.
In this process, you download the CSV template from the analysis software, edit the information, and upload the
completed CSV file for your run.
Prerequisites:
You have the sample information.
To download the CSV template
1. From the dashboard, choose Create New Run.
2. Choose the CSV Upload tab.
3. Choose Download.
The template is saved to the default download directory on your local computer.
4. Open the template, and edit the information, as needed. Ensure that the format of the Adapter, Panel, and Analysis
Workflow information matches that in the analysis software.
5. Save the file.
To upload the CSV file for your run
1. Choose Create New Run.
2. Choose the CSV Upload tab.
3. Drag and drop your edited CSV file to Drop files here, or choose Browse to navigate to the file.
This step can take approximately 10 seconds, depending on the number of samples you upload.
Chapter 3. Create runs
AVENIO ctDNA Analysis Software User Guide, v1.0.0 19
4. Choose Continue.
The Run Settings page appears. This page can take several seconds to appear.
5. Go to Choosing the analysis input file and starting analysis.
Choosing the analysis input file and starting analysis
After you specify your sample entries, you use the Run Settings page to select either Base Call File (BCL) or FASTQ
analysis input files to associate with the run samples, (BCL is the standard output for an Illumina NextSeq Sequencer)
then you start the analysis.
You can convert a BCL file to FASTQ. For instructions, see Converting a BCL output file to FASTQ.
Prerequisites:
A run is created and samples are added.
The admin properly set your User ID (UID) and Group ID (GID) to allow file browsing to the shared
network storage.
The admin configured the correct path to the bcl2fastq executable so that the BCL button is enabled, or you have
FASTQ files already converted from your BCL files.
An Illumina NextSeq was used for sequencing.
To choose a BCL analysis input file
1. On the Run Settings page, choose the file type BCL.
Chapter 3. Create runs
AVENIO ctDNA Analysis Software User Guide, v1.0.0 20
This is the file format that the Illumina NextSeq generates and deposits on your shared network storage for your
environment.
2. For BCL Folder Location, choose Browse to navigate to the available BCL folders.
If the administrator set up the dedicated sequencing input mount, you can select the date of sequencing, machine
name, and Flowcell ID to find the BCL folder. If not, you must know the location of the output files from
sequencing.
The Flowcell ID displayed in the analysis software is taken from the name of the BCL
directory and may
include an extra character.
3. Choose the BCL folder that represents your run, and choose Select.
If the name of the directory is still in the automatic Illumina format, the following run details are displayed:
Date Started
Indicates the date that the sequencer was initiated for that particular sequencing run.
Machine Name
Identification of the Illumina sequencer used for the sequencing run.
Flowcell ID
Unique identifier of the flowcell on which the samples are loaded.
Sequencing
Status of the sequencer run. If you do not see Complete, and you think there is an error,
check the internal contents of the folder to see if the RTAComplete.txt file is present.
If the directory name has changed, only
Sequencing
details are shown.
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Roche AVENIO ctDNA Analysis SW User manual

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