For Research Use Only. Not for use in diagnostic procedures.
Oncomine™ cfDNA Assays
Part III: Variant Analysis
USER GUIDE
for use with:
Oncomine™ Lung cfDNA Assay
Oncomine™ Colon cfDNA Assay
Oncomine™ Breast cfDNA Assay
Catalog Numbers A31149, A31182, A31183
Publication Number MAN0015874
Revision C.0
The information in this guide is subject to change without notice.
DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL,
INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR
USE OF IT.
Revision history: Pub. No. MAN0015874
Revision Date Description
C.0 14 November 2016 Update to include the following Ion Reporter™ workflows:
• Oncomine™ Breast Liquid Biopsy
• Oncomine™ Breast Tumor
B.0 27 October 2016 Update to include the following Ion Reporter™ workflows:
• Oncomine™ Colon Liquid Biopsy
• Oncomine™ Colon Tumor
A.0 29 June 2016 Initial release of variant analysis guide for Oncomine™ Lung Liquid Biopsy
and Oncomine™ Lung Tumor workflows in Ion Reporter™ Software v5.2
and Torrent Variant Caller variant analysis for libraries made with the
Oncomine™ cfDNA Assay kits.
Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept
the terms and conditions of all applicable Limited Use Label Licenses.
Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
©2016 Thermo Fisher Scientific Inc. All rights reserved.
Contents
About this guide ............................................................ 4
■CHAPTER 1 VariantCaller Plugin variant analysis .................... 5
Review Oncomine™ cfDNA assay run results ........................................ 5
■CHAPTER 2 Ion Reporter™ Variant Analysis ........................... 9
Analysis workflows in Ion Reporter™ software ...................................... 9
View results and determine variants of interest ..................................... 9
Export results ................................................................. 13
Oncomine™ annotations ......................................................... 13
Manually launch an analysis ..................................................... 14
■APPENDIX A Documentation and support ............................ 16
Related documentation ......................................................... 16
Obtain information from the Help system ......................................... 16
Customer and technical support ................................................. 16
Limited product warranty ....................................................... 17
Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
3
About this guide
The Oncomine™ cfDNA Assays can be analyzed with the variantCaller plugin in
Torrent Suite™ Software v5.2.1 or in Ion Reporter™ Software v5.2.
For robust detection of low frequency somatic variants from liquid biopsy samples,
we recommend to multiplex up to:
• 8 Oncomine™ Lung cfDNA Assay libraries on a single Ion 530™ chip.
• 6 Oncomine™ Colon cfDNA Assay libraries on a single Ion 530™ chip.
• 12 Oncomine™ Breast cfDNA Assay libraries on a single Ion 530™ chip.
In Torrent Suite™ Software, the Planned Run templates to be used with the
Oncomine™ cfDNA Assays are listed in the following table:
Application Torrent Suite™ Software
template Description
Oncology - Liquid
Biopsy
• Oncomine™ Lung Liquid
Biopsy DNA
• Oncomine™ Colon Liquid
Biopsy DNA
• Oncomine™ Breast Liquid
Biopsy DNA
Planned run template for use
with cell-free DNA (cfDNA)
research samples. Analysis
parameters are optimized for
the sensitive and specific
detection of rare somatic
variants (SNPs, InDels) present
at 0.1% frequency in cfDNA.
• Oncomine™ Lung Tumor
DNA
• Oncomine™ Colon Tumor
DNA
• Oncomine™ Breast Tumor
DNA
Planned run template for use
with solid tumor research
samples from FFPE as well as
fresh frozen tumor tissue.
Analysis parameters are
optimized for the sensitive and
specific detection of rare
somatic variants (SNPs, InDels)
present at 0.5% frequency.
Analysis parameters are
optimized to eliminate false
positives due to DNA damage
resulting from formalin fixation.
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Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
VariantCaller Plugin variant analysis
Review Oncomine™ cfDNA assay run results
The Completed Runs Report from an Oncomine™ cfDNA Assay run is similar to
variantCaller plugin reports. The following outputs have been added.
1. After the run is complete, click Data4Completed Runs and Results, then click
the Run Report for your results.
2. To view a summary of the variant analysis, scroll down to the variantCaller
section, then click the appropriate buon to download variant calls in .vcf or .xls
formats.
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Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
5
3. Review the results in the Median Read Cov, Median Mol Cov, and Targets >
0.8MM columns.
Column Description
Median Read Coverage Reports median coverage across targets.
Median Molecular Coverage reports
median number of individual
interrogated DNA molecules across
targets.
Targets >0.8 Median Molecular Coverage Reports percent of targets with
molecular coverage within 80% of the
median coverage value. This is a new
stricter definition of panel uniformity.
Median Read Coverage and Targets >0.8
Median Molecular Coverage
Measures the quality of the sequencing
run and library performance, while
Median Molecular Coverage measures
the amount and quality of the input DNA
sample.
Median Molecular Coverage Directly influences the limit of detection
in a sample run. We always require two
independent molecular families to
identify a variant for it to be called. Lower
median molecular coverage values result
in less sensitive detection of variants at
0.1% frequency, although still sufficient
for sensitive detection of variants with
higher frequency. For example, Median
Molecular Coverage of 700 is sufficient
for accurate detection of variants at 0.5%
frequency.
For sensitive variant detection down to 0.1% frequency, we see optimal results
when targeting a Median Read Coverage >25,000, Median Molecular Coverage >
2,500, and Targets >0.8 Median Molecular Coverage >60%.
4. Click a Barcode Name of interest to review Variant Calls by Allele.
By default only hotspot alleles calls are shown in the variant table. We do not
report hotspot alleles that did not meet our criteria for calling. However, we do
provide at least one record for each hotspot position. This can include: novel
allele call at hotspot position, hotspot allele call, or absent call when the rst two
are missing.
Chapter 1 VariantCaller Plugin variant analysis
Review Oncomine
™
cfDNA assay run results
1
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Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
Column Description
Frequency Reports the observed frequency of hotspot allele.
LOD Reports limit of detection at hotspot position, which is
based on the number of interrogated DNA molecules
(fragments) containing target.
We use the term 0.1% LOD to mean we have data to
support specific sensitivity and specificity claims (90%
and 98%) at the 0.1% allelic frequency. By default, our
analysis tool uses minimum alternative allele
frequency threshold of 0.05% and we have a technical
lower limit of detection of 0.03% for this method.
Observed frequency can be lower than LOD due to sampling nature of the assay.
If selected to display hotspot positions with absent variant call, then only one
record per hotspot position is displayed and only one of the hotspot alleles at
that position is displayed under "Allele Name".
5. Click View Coverage Metrics to view the total number of interrogated DNA
molecules at hotspot positions (Molecular Coverage), and the number of
molecules containing the variant (Allele Mol Cov).
6. You can modify the types of calls that are displayed in the Allele Calls dropdown
list, by selecting or deselecting Absent, Heterozygous, Homozygous, or No Call.
No calls are variant calls that are classied as systematic errors.
Chapter 1 VariantCaller Plugin variant analysis
Review Oncomine
™
cfDNA assay run results
1
Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
7
7. Select Absent in the Allele Call
dropdown list to visualize hotspot
positions without a valid variant call that
meets our analysis criteria. We report
one record per hotspot position with
missing alternative call, and the
alternative allele is an arbitrary value
distinct from reference. LOD and
molecular coverage metrics at those
positions are measurements for variant
absence among many interrogated
molecules.
8. To view novel alleles, select Novel (sequenced allele that is dierent from the
expected allele dened in the panel hotspot le) in the Allele Source dropdown
list.
Chapter 1 VariantCaller Plugin variant analysis
Review Oncomine
™
cfDNA assay run results
1
8
Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
Ion Reporter™ Variant Analysis
Analysis workflows in Ion Reporter™ software
Analysis workows available in Ion Reporter™ Software include:
Analysis Workflow Description
Oncomine™ Lung/Colon/Breast
Liquid Biopsy - w1.1 - DNA -Single
Sample
Detects and annotates low frequency (to 0.1% limit of
detection) variants (SNPs, InDels) from targeted DNA
libraries from the Oncomine™ cfDNA Assay. This is
compatible with DNA purified from cell-free DNA.
Oncomine™ Lung/Colon/Breast
Tumor - w1.1 - DNA -Single
Sample
Detects and annotates low frequency (to 0.5% limit of
detection) variants (SNPs, InDels) from targeted DNA
libraries from the Oncomine™ cfDNA Assay. Due to
deamination events caused by the FFPE process, the
minimum alternative allele frequency is set to 0.3%.
This makes it compatible with DNA purified from
FFPE tumor tissue as well as fresh frozen tumor
tissue.
In Torrent Suite™ Software, you can plan your run to automatically transfer data to the
appropriate Ion Reporter™ server and be analyzed through one of the Oncomine™
Lung workows.
View results and determine variants of interest
Ion Reporter™ Software analyses are performed automatically on uploading of the
data les from the Torrent Suite™ Software. To view the results:
1. Log in to the Ion Reporter™ Software.
2. Click the Analyses tab.
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Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
9
3. To view Oncomine™ cfDNA Assay results, select Oncology-Liquid Biopsy in the
Application dropdown list.
The resulting Analyses screen displays the Oncology-Liquid Biopsy analyses.
4. In the Analyses screen you can:
To… Action
Open an Analysis
Results screen.
Click the hyperlink (in the Analysis column).
View details Click in the blank space of the analysis' row.
Sort chronologically Click the Created On column header.
1 32
1Open Analysis Results screen
2View details
3Sort chronologically
5. In the Analysis Results screen sort or
lter the data using the Oncomine™-
specic annotations. See the software
help menu for more options.
In the Filter Chains panel, notice that the
Oncomine Variants (5.2) lter chain has
been applied by default.
Chapter 2 Ion Reporter™ Variant Analysis
View results and determine variants of interest
2
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Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
6. In the Liquid Biopsy column, view Mol Depth, Mol Counts, and other columns.
Column Description
Molecular Depth Reports number of interrogated DNA molecules containing
target. It defines limit of detection at hotspot position in a
particular run and sample. For instance, if molecular depth is
≥1,500, you can have high confidence that no variant is present
at 0.2%. If molecular depth is ≥2,500, you can have high
confidence that no variant is present down to 0.1% LOD.
For reference calls, Molecular Depth provides measurable
metric that serves as confirmation for variant absence among
a large number of interrogated molecules.
Molecular Counts Reports the number of detected DNA molecules containing
variant allele.
Chapter 2 Ion Reporter™ Variant Analysis
View results and determine variants of interest
2
Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
11
7. In the Oncomine tab, click the column headers to sort the list of variants by
Oncomine Variant Class and Oncomine Gene Class.
Reference calls display chromosomal position with empty value in amino acid
change eld.
8. In the Ontologies tab, click the column headers to sort the list by variant Type or
Genes to analyze your results.
Chapter 2 Ion Reporter™ Variant Analysis
View results and determine variants of interest
2
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Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
Export results
To export a report:
1. Click Download, then select All Variants, Filtered Variants or Current Results
TSV.
2. Click Home4Notications to open the Notications screen, then click to
download your results.
The software generates a .zip le with three folders: QC, Variants, and
Workow_Seings. Within the Variants folder, you’ll nd the Oncomine™
annotated .vcf le, which is used by the Oncomine™ Knowledgebase Reporter.
3. Open the annotated .vcf le, then scroll to the Oncomine™ annotations.
Oncomine™ annotations
Variant Type Oncomine™
Gene Class
Oncomine™
Variant Class Annotation Criteria
Gain of
Function
Missense
Hotspot
Mutation
Gain-of-
Function
Hotspot • Variant's functional impact is missense
• Variant occurs in Gain of Function gene
• Variant's transcript and codon position
occur in predefined missense hotspot
list
Gain of
Function In
Frame
Hotspot
Mutation
Gain-of-
Function
Hotspot • Variant occurs in Gain of Function gene
• Variant's function, transcript and
coding syntax occur in pre-defined in-
frame hotspot list
Loss of
Function
Missense
Hotspot
Mutation
Loss-of-
Function
Hotspot • Variant's functional impact is missense
• Variant occurs in Loss of Function gene
• Variant's transcript and codon position
occur in predefined missense hotspot
list
Chapter 2 Ion Reporter™ Variant Analysis
Export results
2
Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
13
Variant Type Oncomine™
Gene Class
Oncomine™
Variant Class Annotation Criteria
Loss of
Function In
Frame
Hotspot
Mutation
Loss-of-
Function
Hotspot • Variant occurs in Loss of Function gene
• Variant's function, transcript and
coding syntax occur in pre-defined in-
frame hotspot list
Gain of
Function
Splice Site
Hotspot
Mutation
Gain-of-
Function
Hotspot • Variant occurs in Gain of Function gene
• Variant's transcript, location, and exon
occur in pre-defined splice site hotspot
list
Loss of
Function
Synonymous
Hotspot
Mutation
Loss-of-
Function
Hotspot • Variant occurs in Loss of Function gene
• Variant's function, transcript, and
coding syntax occur in pre-defined
synonymous hotspot list
Manually launch an analysis
If your analysis did not automatically launch, you can launch it manually.
1. Click the Workows tab (or Analyses tab).
2. In the Application dropdown menu, lter for Oncology-Liquid Biopsy.
3. In the Workow Name column, click on the workow appropriate to your assay.
•Oncomine™ Lung/Colon/Breast Tumor - w 1.1 - DNA - Single Sample or
•Oncomine™ Lung/Colon/Breast Liquid Biopsy - w 1.1 - DNA - Single
Sample
4. Select Launch Analysis in the Actions dropdown.
5. Select one DNA sample.
a. Search by any unique identier you used to label the samples during setup.
b. Click Add Samples.
Chapter 2 Ion Reporter™ Variant Analysis
Manually launch an analysis
2
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Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
The samples populate a eld on the right side of the screen.
6. Click Next.
7. Conrm that the Oncomine Variant Annotator v2.1 plugin is selected, then click
Next.
8. (Optional) Modify the name, then add a description.
9. Click Launch Analysis.
10. Follow the steps in the “View results and determine variants of interest“ on
page 9 and “Export results“ on page 13 of this guide to sort, lter, and generate
reports of your results.
Chapter 2 Ion Reporter™ Variant Analysis
Manually launch an analysis
2
Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
15
Documentation and support
Related documentation
Document Description
Oncomine
™
cfDNA Assays, Part I: Library
Preparation User Guide
(Pub. No. MAN0014688)
Describes the preparation of Oncomine™
cfDNA Assay libraries.
Oncomine
™
cfDNA Assays, Part II: Plan a
Run, Template Preparation, and
Sequencing User Guide
(Pub. No. MAN0015873)
Describes the automated template
preparation of Oncomine™ cfDNA Assay
libraries using the Ion Chef™ System for
sequencing on the Ion S5™ System.
Oncomine
™
cfDNA Assays, Part III: Variant
Analysis User Guide
(Pub. No. MAN0015874)
Describes how to perform variant analysis
on Oncomine™ cfDNA Assay sequence data,
and how to view, sort, and filter results.
Note: For additional documentation, see “Customer and technical support“ on
page 16.
Obtain information from the Help system
The Torrent Suite™ Software has a Help system that describes how to use each feature
of the user interface.
In the toolbar of the Torrent Suite™ Software window, click Help4Software Help.
You can use the Help system to nd topics of interest by:
• Reviewing the table of contents
• Searching for a specic topic
Customer and technical support
Visit thermosher.com/support for the latest in services and support, including:
• Worldwide contact telephone numbers
• Product support, including:
– Product FAQs
– Software, patches, and updates
– Training for many applications and instruments
• Order and web support
A
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Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
• Product documentation, including:
– User guides, manuals, and protocols
–Certicates of Analysis
– Safety Data Sheets (SDSs; also known as MSDSs)
Note: For SDSs for reagents and chemicals from other manufacturers,
contact the manufacturer.
Limited product warranty
Life Technologies Corporation and/or its aliate(s) warrant their products as set forth
in the Life Technologies' General Terms and Conditions of Sale found on Life
Technologies' website at www.thermosher.com/us/en/home/global/
terms-and-conditions.html. If you have any questions, please contact Life
Technologies at www.thermosher.com/support.
Appendix A Documentation and support
Limited product warranty
A
Oncomine
™
cfDNA Assays, Part III: Variant Analysis User Guide
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