Thermo Fisher Scientific Oncomine cfDNA Assays Part III: Variant Analysis User guide

Brand: Thermo Fisher Scientific

Model: Oncomine cfDNA Assays Part III: Variant Analysis

Type: User guide

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.

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

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.

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 buon to download variant calls in .vcf or .xls

formats.

Oncomine

™

cfDNA Assays, Part III: Variant Analysis User Guide

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

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 classied as systematic errors.

Chapter 1 VariantCaller Plugin variant analysis

Review Oncomine

™

cfDNA assay run results

Oncomine

™

cfDNA Assays, Part III: Variant Analysis User Guide

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 dierent from the

expected allele dened in the panel hotspot le) in the Allele Source dropdown

list.

Chapter 1 VariantCaller Plugin variant analysis

Review Oncomine

™

cfDNA assay run results

Oncomine

™

cfDNA Assays, Part III: Variant Analysis User Guide

Ion Reporter™ Variant Analysis

Analysis workﬂows in Ion Reporter™ software

Analysis workows 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 workows.

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.

Oncomine

™

cfDNA Assays, Part III: Variant Analysis User Guide

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™-

specic 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

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

Oncomine

™

cfDNA Assays, Part III: Variant Analysis User Guide

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

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 Home4Notications to open the Notications screen, then click to

download your results.

The software generates a .zip le with three folders: QC, Variants, and

Workow_Seings. 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

Oncomine

™

cfDNA Assays, Part III: Variant Analysis User Guide

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 Workows tab (or Analyses tab).

2. In the Application dropdown menu, lter for Oncology-Liquid Biopsy.

3. In the Workow Name column, click on the workow 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 identier you used to label the samples during setup.

b. Click Add Samples.

Chapter 2 Ion Reporter™ Variant Analysis

Manually launch an analysis

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. Conrm 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

Oncomine

™

cfDNA Assays, Part III: Variant Analysis User Guide

Documentation and support

Thermo Fisher Scientific Oncomine cfDNA Assays Part III: Variant Analysis User guide

Thermo Fisher Scientific Oncomine cfDNA Assays Part III: Variant Analysis User guide

Thermo Fisher Scientific Oncomine cfDNA Assays Part III: Variant Analysis User guide

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