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Thermo Fisher Scientific Oncomine Focus Assay, Part V: Variant Analysis User guide

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For Research Use Only. Not for use in diagnostic procedures.
Oncomine™ Focus Assay, Part V:
Variant Analysis
USER GUIDE
Publication Number MAN0013549
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
Table 1 Revision history of MAN0013549
Revision Date Description
C.0 29 January 2016 Added information about Torrent Suite™ Assay
Development Software
B.0 16 October 2015 Revised document. New features in Ion Reporter™
Software v5.0 included.
A.0 27 May 2015 New document
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 | 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
■CHAPTER 1 Methods ..................................................... 5
Analysis workflows in Ion Reporter™ Software ...................................... 5
View results .................................................................... 6
Export Results .................................................................. 8
Launch an Oncomine™ Analysis in Ion Reporter™ Software ............................ 9
(Optional) Import sample VCF files and add Oncomine™ Focus annotations ............ 11
■APPENDIX A CNV Baseline Creation .................................. 17
Use new VCIB CNV Baseline ..................................................... 17
Create a CNV Baseline .......................................................... 17
Augment (Add Samples to) an Existing VCIB CNV Baseline .......................... 20
Create an Ion Reporter™ Analysis Workflow ....................................... 22
Launch an Analysis ............................................................. 24
■APPENDIX B Subset Filter Creation .................................. 27
Create a Gene-level Filter ....................................................... 27
Create a Variant-level filter ...................................................... 29
Create a new VariantDB using the provided file ................................ 29
Create a new Annotation Set using the new VariantDB and existing
Oncomine™ annotation sources .............................................. 30
Create a new Filter Chain using the new VariantDB ............................ 32
Create a copied Workflow using both the new Annotation Set and new
Filter Chain ............................................................... 33
Use new workflow ......................................................... 34
■APPENDIX C CNV Somatic Confidence Filter ........................ 35
Understanding CNV Somatic Confidence Range .................................... 35
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
3
■Documentation and support ............................................. 37
Related documentation ......................................................... 37
Obtaining information from the Help system ....................................... 37
Customer and technical support ................................................. 38
Limited product warranty ....................................................... 38
Contents
4
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
Methods
Analysis workflows in Ion Reporter™ Software
To analyze your Oncomine™ Focus Assay data using Ion Reporter™ Software:
• In Torrent Suite™ Software, if your Planned Runs were set up correctly,
automated analysis has already been performed and you can view the
Oncomine™ analysis results in Ion Reporter™ Software.
• In Torrent Suite™ Assay Development Software, you can import the run data into
your local Ion Reporter™ server for further analysis.[1] For detailed instructions,
see the latest Ion Reporter™ Software User Guide, available on the Ion Community at
the following link: ioncommunity.thermosher.com/docs/DOC-9594.
Note: For instructions on manually launching an analysis, see “Launch an
Oncomine™ Analysis in Ion Reporter™ Software“ on page 9.
Note: Microsoft™ Excel™ is required for viewing VCF, CSV and TSV les.
Available workows in Ion Reporter™ Software include:
1
[1] Ion PGM™ Dx System with Torrent Suite™ Assay Development Software is For Research Use
Only. Not for use in diagnostic procedures.
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
5
Analysis Workflow Description
Oncomine™ Focus v2.0 - DNA and Fusions -
Single Sample
Detects and annotates low frequency
somatic variants (SNPs, InDels, CNVs) from
targeted DNA libraries, as well as gene
fusions from targeted RNA libraries, of the
Oncomine™ Focus Assay.
Oncomine™ Focus v2.0 - DNA - Single
Sample
Detects and annotates low frequency
somatic variants (SNPs, InDels, CNVs) from
targeted DNA libraries of the Oncomine™
Focus Assay.
Oncomine™ Focus v2.0 - Fusions - Single
Sample
Detects and annotates gene fusions from
targeted RNA libraries of the Oncomine™
Focus Assay.
Oncomine™ Focus v2.0 - Annotate Variants -
Single Sample
Annotates VCF files from the Oncomine™
Focus Assay.
View results
1. Log into Ion Reporter™ Software.
2. Click the Analyses tab.
3. In the Application drop-down menu, select the type of analysis you want to
focus on. The Oncomine-specic analyses are: DNA, Fusions, DNA and Fusions,
and Annotate Variants. (For this example, we have selected DNA and Fusions.)
The resulting Analyses grid displays the DNA and Fusions analyses.
Chapter 1 Methods
View results
1
6
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
4. Here, you can sort results in many ways, including:
• Sort the analyses chronologically in the Created On column.
• View details of an analysis, click on the blank space in the analysis’ row.
• Click on the sample’s hyperlink (in the Analysis column) to open its variant
review grid.
5. You can also use a variety of approaches for sorting and ltering data. Use of the
Oncomine™-specic annotations are described here. See software's help menu for
more options.
a. Click on the hyperlink in the Analysis column to view the Analysis Results
variant review grid.
b. In the Filter Chains panel on the right, notice that the Oncomine Variants,
5% CI CNV ploidy >= gain of 2 over normal (5.0) lter chain has been
applied by default. This lter chain removes variants that do not match this
lter. For more information on this Filter Chain option, please see Appendix:
Understanding CNV Somatic Condence Range.
Chapter 1 Methods
View results
1
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
7
6. On the Oncomine tab, you can sort variants in the Oncomine Variant Class and
Oncomine Gene Class columns.
7. Click on the Ontologies tab. Here you can sort variants by Type or by Genes.
Export Results
Once you have determined your variants of interest, you can export a report.
1. Click the Download buon. Choose All Variants, Filtered Variants or Current
Results TSV. (For this example, we chose Filtered Variants.)
The software generates a zipped le with four folders: QC, Variants,
Workow_Seings, and CNVBaseline. Within the Variants folder, you’ll nd the
Oncomine™ annotated VCF le, which is used by the Oncomine™
Knowledgebase Reporter. For more information on the CNV output les, see the
CNV Baseline Creation appendix.
Chapter 1 Methods
Export Results
1
8
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
2. Scroll through the annotated VCF le and you can see where Oncomine™
annotations have been added.
The following annotations may be added:
Oncomine™ Gene Class Oncomine™ Variant Class Class Membership
Gain of Function Hotspot Hotspot missense or in-
frame mutation in one of
the 35 Oncomine™ Focus
hotspot genes (hotspot =
mutated more often than
would be expected by
random chance in tumor
samples in the Oncomine™
Knowledgebase).
Gain of Function Amplification Amplification (5%
Confidence Interval CNV
ploidy >= gain of 2 over
normal) in one of the 19
Oncomine™ Focus copy
gain genes.
Gain of Function Fusion Positive fusion call in one
of the 271 Oncomine™
Focus fusion variants.
For detailed information about Copy Number Variation algorithms, see Copy
Number Variation (CNV) Algorithm Description, available on Ion Community,
DOC-8764.
Launch an Oncomine™ Analysis in Ion Reporter™ Software
1. Go to Workows tab (or Analyses tab).
2. In the Application drop-down, lter for DNA and Fusions.
3. Enter Oncomine in the search box.
Chapter 1 Methods
Launch an Oncomine™ Analysis in Ion Reporter™ Software
1
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
9
4. Click on the Oncomine™ Focus v2.0-DNA and Fusions-Single Sample workow
in the Workow Name column.
5. Click the Actions drop-down menu and select Launch Analysis.
6. Pick one DNA sample and one RNA (Fusions) sample. Search by any unique
identier you used to label the samples during setup. Make sure the samples
have had percent cellularity, sample type and cancer type dened.
7. Click Add Samples. The samples populate a eld on the right side of the screen.
8. Provide a group name.
9. Click the Add to Analysis buon. The screen displays a "Ready to Analyze"
message. Click Next.
Chapter 1 Methods
Launch an Oncomine™ Analysis in Ion Reporter™ Software
1
10
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
10. Conrm that the Oncomine Variant Annotator v2.0 plugin is selected by default.
11. Click Next. The screen displays an "Analysis ready to launch" message.
12. (Optional) Modify the name and add a description.
13. Click Launch Analysis.
14. Follow the steps in the View Results and Export Results sections of this guide to
sort and lter your results and generate reports.
(Optional) Import sample VCF files and add Oncomine™ Focus
annotations
The Oncomine™ Variant Annotator plugin allows you to manually import samples,
apply Oncomine™ annotations, and generate a VCF le that includes these
annotations.
1. On the Sample page, click the Dene Sample buon and select either Manual or
Import from CSV option. (For this example, we selected Manual.)
Chapter 1 Methods
(Optional) Import sample VCF files and add Oncomine™ Focus annotations
1
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
11
2. On the Dene Samples screen, select the le type column that corresponds with
the le type you want to upload. (For our example, BAM and VCF columns are
shown. We clicked the VCF column, and then clicked the Upload a VCF buon.)
3. Select the le you want to upload and click Upload VCF. The software uploads
and validates the le. Click Close.
4. Select the newly uploaded le and add it to the sample denition.
a. Place a checkmark next to le name.
b. Click the Add to Sample buon (vertical text).
c. Name it. (For our example, demo-sample-vcf.)
Chapter 1 Methods
(Optional) Import sample VCF files and add Oncomine™ Focus annotations
1
12
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
d. Click the Add to Sample List buon, click Next.
The Aributes page launches.
5. Click the Add Aribute buon if you want to add information. Otherwise, click
Next.
6. The system returns you to the Samples page. Check that your new samples are
now on the list. Click Save.
7. Click the Analyses tab.
a. Click the Launch Analysis buon and select Manual or Batch. (For our
example, we selected Manual.)
The Launch Analysis screen appears.
Chapter 1 Methods
(Optional) Import sample VCF files and add Oncomine™ Focus annotations
1
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
13
8. In the Workow column, select the Oncomine™ Focus v2.0 - Annotate Variants -
Single Sample. Click Next.
The Samples screen shows you all of the samples this workow can be run on.
The “eye” symbol denotes samples that have already had this workow run on
them.
9. Select the sample you want to run through the workow by placing a checkmark
next to it, and click Next.
10. Conrm that the Oncomine™ Variant Annotator v2.0 plugin is selected by
default. Click Next.
Chapter 1 Methods
(Optional) Import sample VCF files and add Oncomine™ Focus annotations
1
14
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
11. Click Launch Analysis buon. This step may take up to 10 minutes. The
software is adding Oncomine™ annotations to variants which have already been
algorithmically detected by the variant caller algorithm and are present in the
VCF le. For the other three workows, variant caller, mapping, and annotation
algorithms are applied to the sample, followed by the adding of Oncomine™
annotations to the VCF.
12. From the Analysis Results page, you can lter your results. On the right side of
the screen, you’ll nd Filter Options. (In this example, we found 3 variants.)
13. In the Filter Chains panel, notice that Oncomine Variants, 5% CI CNV ploidy >=
gain of 2 over normal (5.0) has been applied by default. This lter chain changes
the table so that only the Oncomine™variants are shown.
14. At this point in time, you can download a results zip bundle which includes an
annotated VCF le and other analysis results les.
15. From the Download buon, you can choose All Variants or Filtered Variants. (For
our example, we chose Filtered Variants.)
16. The system generates a .zip le. Download and unzip the le.
The content includes four folders: QC, Variants, Workow Seings, and
CNVBaseline. Within the Variants folder you will nd the Oncomine™ annotated
VCF le which is used by the Oncomine™ Knowledgebase Reporter.
Chapter 1 Methods
(Optional) Import sample VCF files and add Oncomine™ Focus annotations
1
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
15
17. Scroll through the annotated .vcf le and you can nd where the Oncomine™
annotations have been added.
Chapter 1 Methods
(Optional) Import sample VCF files and add Oncomine™ Focus annotations
1
16
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
CNV Baseline Creation
In AmpliSeq™ assays, Copy Number estimates are made by counting reads for each
amplicon, making adjustments to account for certain types of variability, comparing
those read counts to expected counts for those amplicons in a "normal" sample, and
then making further adjustment.
Known sources of variability include pool imbalance (when the assay has more than
one pool of amplicons), total number of reads and per amplicon aributes of GC
proportion, and length of the amplicon insert. In practice, we observe other variability
that does not associate with known aributes yet is systematic. The method we use
trains on a large number of diverse samples, captures systematic eects, and encodes
these into a le (the "baseline").
When augmenting a baseline, new samples are run, the size of each systematic eect
encoded in the baseline is estimated, and a correction is applied to remove the eect.
These added samples need not be normal, and should be diverse so as to capture
likely systematic variation.
The following instructions walk you through using the new Variability Correction
Information Baseline (VCIB) CNV baseline, creating a new VCIB CNV baseline, or
augmenting an existing VCIB CNV baseline for Oncomine™ Focus Assay panels.
Use new VCIB CNV Baseline
If you want to use the new VCIB CNV baseline included in Ion Reporter™ Software
v5.0, simply select it when creating your workow. See Create an Ion Reporter™ CNV
Workow section for details.
Create a CNV Baseline
Ion Reporter™ Software provides a wizard to guide you through Copy Number
Variation (CNV) Baseline creation.
1. Click the Workows tab, then click the Presets sub-tab.
A
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
17
2. On the Create Preset menu, select Copy Number Baseline.
3. Select AmpliSeq.
4. Select Oncomine Focus Panel v1.1 Regions as your Targets Region le. Click
Next.
Appendix A CNV Baseline Creation
Create a CNV Baseline
A
18
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
5. On the Algorithm Type page, select CNV VCIB 1.0 for the latest CNV algorithm
to be applied. Click Next.
6. Select at least 48 samples and ag at least 6 of these "Normal" and click Next.
Male or Female gender must be specied for Normal samples, but samples not
agged as normal can be male, female or unknown. You can use the Summary
panel to see your totals.
7. Enter a name for your baseline and then click Create Baseline to save it.
Note: Logs for both successful and failed analyses include the
"BaselineCreation.log" le which has the BAM les named that were rejected due
to similarity to other les in the baseline, as well as the
"map.TmapMergeActor-00.err" le which has the BAM les named which were
rejected due to QC failure.
Next, proceed to the Create an Ion Reporter CNV Workow section where you will
add this new baseline to your workow.
Appendix A CNV Baseline Creation
Create a CNV Baseline
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Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
19
Augment (Add Samples to) an Existing VCIB CNV Baseline
Ion Reporter™ Software provides a wizard to guide you through Copy Number
Variation (CNV) Baseline creation. This example describes how to add additional
samples to an existing CNV baseline.
1. Click the Workows tab, then click the Presets sub-tab.
2. On the Create Preset menu, select Copy Number Baseline.
3. Select AmpliSeq.
4. Select Oncomine Focus Panel v1.1 Regions as your Targets Region le. Click
Next.
5. On the Algorithm Type page, verify CNV VCIB 1.0 is selected.
Appendix A CNV Baseline Creation
Augment (Add Samples to) an Existing VCIB CNV Baseline
A
20
Oncomine™ Focus Assay, Part V: Variant Analysis User Guide
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