Interpreting Variants in Clinical Reporter

Charlene Son-Rigby -

Clinical Grade and Regions of Homozygosity

Select Variants for Solo Report without VAAST Test Workflow

Scoring and Classifying Variants

Interpreting Trio or Quad Tests

Interpreting Duo Tests

Using Phevor to Leverage Phenotype in your Solo or Family Interpretation

Confirming Variants via Sanger Sequencing

 

Opal Clinical provides multiple tools to accelerate and standardize your interpretation process. These include tools to quickly assess evidence and classify variants, re-use classifications for variants seen in previous patients, and prioritization and ranking tools so the most likely candidates are reviewed first. This Interpreting Variants in Clinical Reporter section of the User Guide describes these tools.

Once a test has been set up, the variants identified in the test can be interpreted.

On the Clinical Reports page, click on the Actions dropdown menu of the desired report and select “Review Results”. This will launch the variant interpretation and reporting workflow.

The Variant Table columns and all Filter options on the Variant Selection and Variant Interpretation pages are described in detail in the articles SNVs and Indels and Structural Variants

 

Omicia Clinical Grade and Regions of Homozygosity

Omicia Clinical Grade

Before starting an interpretation it’s important to confirm the quality of your sequence data. You can do this with the Omicia Clinical Grade. The Omicia Clinical Grade is a compilation of commonly used criteria for evaluating the quality of genomes and exomes. Click the signal-strength bar icon next to genome name at the top left of the Select Variants page.

See Appendix 4 for more information on Clinical Grade Score.

 

Regions of Homozygosity

You may also want to review regions of homozygosity within a sample. Opal’s Regions of Homozygosity (ROH) viewer is designed to highlight whether your genome contains any large (>1 MB) regions of homozygosity. Such regions could represent biological features such as consanguinity, uniparental disomy, or large deletions. It will also pick up on artifacts of VCF generation that could merit further investigation.

It displays information about large Runs of Homozygosity in two forms:

  • A graphic, displaying the fraction of homozygous variants along the length of all chromosomes. This is a direct visualization of the information present in your uploaded VCF.
  • A table, displaying calculated Runs of Homozygosity. Opal can display regions which are at least 1 MB, 3 MB or 5MB in size with a density of homozygous variants of at least 75%.  The 75% is set to allow for sequencing errors. The precise start and end points of the runs are approximate only.

Select Variants for Solo Report without VAAST Test Workflow

All workflows will have a set of candidate variants in the Interpret Variants page except for the Solo Report without VAAST workflow. For this workflow, the first step is to select variants:

  1. On the Clinical Reports page, click on the Actions dropdown menu of the desired report and select “Review Results”.
  2. You will be presented with a clinical version of Variant Miner, which allows you to use Variant Miner’s rich filtering capabilities to select variants for your clinical exome interpretation. For more information on using Variant Miner’s filters, please see the Variant Miner section of the User Guide.
  3. Click on a variant in the Variant Miner table to highlight it in blue.
  4. Click on the “Add Variant to Report” button on the upper right to include highlighted variants in your interpretation. You should think of this as creating a short list of candidate variants In the Interpret Variants page, you will review this short list of variants in detail and decide on which variants to report.
  5. Click the “Return to Report” button to go to the Interpret Variants page.

You can move back and forth between Clinical Variant Miner and Interpret Variants to add additional variants to your short list. In Interpret Variants, click the “Add Variants” button to return to Clinical Variant Miner.

 

 

Beacon

When a variant has not been observed in any of the reference populations, a beacon icon will appear in the allele frequency field in the Interpret Variants table. Beacon is a project launched by the Global Alliance for Genomics and Health (GA4GH). Beacon allows labs to share de-identified information at the variant level to determine whether they have patients with the same variant. A number of reference sites including NCBI, Sanger, the Institute of Systems Biology and UCSC have launched public web services. Clicking on the beacon icon in Opal will send a query to each of the Beacon reference sites asking whether they have any genomes. The only information exchanged is the chromosome, position and variant.

More information on Beacon is available at the GA4GH Beacon project site.

Gene Summary and Gene Viewer

Clicking on a gene symbol will display the Gene Summary window. This summary includes a graphical representation of the gene, information on the gene from NCBI Entrez with external links to USCS Genome Browser, Ensembl, and if applicable, to GeneTests, the NCBI Genetics Reference, and CIViC.

You can zoom in to a specific region in a gene by clicking and holding the mouse on one side of the region, and dragging to include the complete region you want to focus on. When you release the mouse, the gene viewer will update to display only this region. Unzoom with the magnifying glass at the bottom of the gene viewer. Use the arrow buttons to  allowing you to navigate upstream and downstream in the gene. the gene more easily.

The Gene Variants tab provides a list of all gene variants that met the initial filtering criteria for this report, and are presented on a transcript basis with the HGVS nucleotide and protein nomenclature. Clicking on a variant will highlight its location in the gene viewer above.

The Evidence tracks display vertical lines representing variants annotated in the various databases. If you mouse over a line, a hand pointer will appear. Clicking the mouse will present a dialog with the evidence and a link to the source database, if available.

Repeat Track annotations are from the Ensembl 75 Homo Sapiens Core Database, from the repeat_feature and repeat_consensus tables.

NLP Phenotype Mapper

NLP Phenotype Mapper generates a list of conditions associated with a gene using natural language processing of PubMed abstracts. NLP provides a ranked list of conditions, strength of association and prioritized PubMed abstracts. The PubMed abstracts are ordered based on relevance.

Scoring and Classifying Variants

This section describes Opal Clinical's variant scoring and classification features.

These features have been optimized for labs that want to standardize on the ACMG classification methodology. However, labs that want to evaluate evidence and classify variants without capturing their detailed decision making process can easily use the same Opal interface, and will benefit from expanded gene and variant annotations, broader support for condition-gene curation, and more robust citation management for segregation and functional studies.

In addition, the Opal architecture is flexible, allowing customization of classification rules, enabling labs with existing classification systems, as well as those that have extended the ACMG criteria to further standardize and accelerate their interpretation processes. If you are interested in customizing classification rules, please contact support@fabricgenomics.com.

 

Mouse over a variant in the Interpret Variants table, and a "Score Variant" tab will appear under the Gene symbol. (Note: Previously Scored Variants will display a "Review Scoring History" tab, Scored Variants will display a "View Scoring").

Click on the "Score Variant" tab to open the Score Variant detail view. The Score Variant detail view is displayed within the Interpret Variants table, underneath the variant you selected. This Score Variant detail view replaces the Interpret Variants modal, and provides detailed information on the variant and gene, as well as allowing you to score and classify the variant. 

The Score Variant detail view has five tabs:

Tab Description
Scoring Score variant using the 2015 ACMG Guidelines, and Set the Classification. For more information about the guidelines, see the following article:  https://www.acmg.net/docs/Standards_Guidelines_for_the_Interpretation_of_Sequence_Variants.pdf
Scoring History Review previous Scores for this variant, as well as its ClinVar classification history. Also review classifications of other variants in this gene from the Classified Variant Database
Citations Review and manage citations. Citations are automatically pre-populated by Opal Annotation engine, and you can manually add citations. Citations can be curated as cosegregation or functional study evidence. 
Condition-Gene Review condition - gene associations curated in your lab Workspace, from ClinVar and OMIM, and from Fabric Genomics' Natural Language Processing (NLP) Phenotype Mapper
Gene Knowledge Base Review gene and variation spectrum data to understand mechanisms for disease in this gene

 

These tabs are explained in more detail in the following sections.

The main workflow is on the Scoring tab, where you evaluate and score a variant. You can curate information in the Condition-Gene, Citations and Variant Description and Notes Tabs. All of the information you curate will be available in your workspace when the variant or gene is seen again.

Condition-Gene Tab

If no Condition-Gene Associations have been associated with this variant in your lab's Workspace, the first tab you will see in the Score Variant detail view is the Condition-Gene tab. 

Select or create a Condition-Gene Association to begin the scoring and classification process. 

The Condition-Gene Associations tab displays condition-gene associations from ClinVar and OMIM, as well as Fabric Genomics' NLP Phenotype Mapper:

  • Workspace Condition-Genes: select an existing Condition-Gene or create a new one using the "+New Condition-Gene" button
  • ClinVar and OMIM Condition-Genes: ClinVar and OMIM associations can be copied and edited using the "Copy to Workspace" link. 
  • NLP Phenotype Mapper: Review associated conditions identified in PubMed abstracts, then create a new Workspace Condition-Gene association.

 

New Workspace Condition-Gene associations are saved in the current workspace, so the data is automatically available for future clinical interpretations. These Workspace associations can be also be viewed and updated in the Condition-Gene Association page, accessible from the Home page.

Scoring Tab

Once you have selected a Condition-Gene Association, you can score and classify the variant in the Score Variant tab. The Scoring tab presents the following information:

  • Scoring Criteria: Explanation of the ACMG criterion being evaluated
  • Scoring Tools: Tools and information within Opal to support evaluation of the criterion. The "?" icon provides additional mouse-over help. The Scoring Tools section may display data or direct you to a different tab to evaluate information
  • Criteria Met? Evaluation of the ACMG criterion. A simple yes/no evaluation, or in the case of co-segregation a strong/moderate/supporting/none evaluation
  • Scoring Summary: automatically generated text describing the evaluation of the ACMG criteria for this variant

 

When you enter an answer in the Criteria Met? section, the answer is saved and you are presented with the next criterion. As you evaluate the individual criterion, the Inferred Class for the variant is updated. The Inferred Class is shown in bold blue at the top of the Score Variant detail tab.

Several of the 28 ACMG criteria can be answered automatically based on allele frequency or variant effect, accelerating and streamlining classification. Automatically entered answers are highlighted with a flag, and can be overridden. If auto-answering is not enabled in your workspace, please contact support@fabricgenomics.com
 
In addition, if you answer Yes to some criteria, Opal will automatically enter "No" for criteria that is mutually exclusive.  Again, automatically entered answers are highlighted with a flag, and can be overridden. 

 

Variant Description, Scoring Summary, Internal Notes and Variant History

  • Variant Description: capture information on the variant and rationale for variant classification for inclusion in the Clinical Report. This information in this field will be saved in Classified Variant Database, and available for immediate reuse when the variant is seen in other patients. Do not include add patient specific information in this field. The contents of this field will appear in the Summary Findings field on the Review Report page, and patient-specific information can be added in the Review Report page.
  • Scoring Summary: automatically generated text based on assessment of ACMG classification criteria. All or some of this text can be copied into the variant description, as desired.
  • Internal Notes: track progress and communicate with interpretation team members. Each note is versioned with author and time stamp. Internal Notes cannot be edited due to audit requirements.
  • Variant History: Historical log of evaluation of criteria and applied classification 

The footer of the Scoring tab provides these functions:

  • Add Internal Note: button to switch to Internal Notes tab (see previous section)
  • Show Scoring Sheet button: view a summary of the evaluation of the ACMG criteria for this variant
  • Set Classification button: set the class for this variant

 

Scoring History Tab

If this variant or another variant in this gene was classified within an Approved Clinical Report in previous patients within your institution, this history will be displayed in the Scoring History Tab. In addition, if this variant was submitted to ClinVar, this history will be provided.

The Your Scoring History for this Gene table provides information on the classifications for other variants in this gene. Data is presented as a variation spectrum by classification. Only SNVs are currently supported. 

If a variant was previously Scored, you will see information in the Your Scoring History for this Variant table. Using the Actions drop-down menu, you can:

  • Accept Scoring: applies the previous score for the variant to the current report
  • Use as Starting Point: pre-populates the ACMG criteria evaluation
  • Show Scoring Sheet: displays the summary of the evaluation of the ACMG criteria from the previously scored variant

You can also review any classifications from ClinVar in the ClinVar Classification History for this Variant table.

 

Citations Tab

In the Citations tab, you can review and manage literature evidence. Citations are automatically pre-populated by Opal Annotation engine, and you can manually add citations. Citations can be curated as cosegregation or functional study evidence. 

You can also search for citations on Google or Google Scholar using the text field in this tab. 

Citations from Opal's Variant Annotation: citations automatically populated from Opal's annotation pipeline can be curated as Functional or Cosegregation Studies

For cosegregation studies, you can capture phenotype, the number of affected and unaffected with and without the variant, as well as the overall assessment of the evidence the study. For functional studies, you can capture the type of experiment, assess the methodology and controls, and provide an overall assessment.

If your lab has an internal server to manage literature resources, you can also save the URL to the saved paper to simplify access for future review. 

Any citations you associate with a reported variant can be automatically included in the References section of the Clinical Report.

 

Gene Knowledge Base Tab

The Gene Knowledge Base provides information on known mechanisms for disease within this gene based on data in ClinVar and ExAC, as well as calculations of probability of loss-of-function intolerance (pLI) and missense z-scores (Somocha et al).

The Gene Knowledge Base complements the Gene Viewer and Summary accessible when clicking on the Gene symbol hyperlink for your variant in the Interpret Variants table.

 

Variant Description and Notes Tab

The Variant Description and Notes tab contains 3 collapsable fields.

The Variant Description for Report field can be populated with text that can be reused when this Scored variant is seen in future patients. The Variant Description is intended to be included in the Clinical Report, and will be displayed in the Summary Findings on the Review Report page.

Internal Notes allow you to track progress or communicate with interpretation team members. Each note is versioned with author and time stamp. 

Variant History allows you to review the history of criteria selection and classification for this scoring instance.

 

Mark as Scored and Specify Report Section

Once all required variants in your panel or test have been reviewed and classified, use the Bulk Update function to mark all of them as Scored. This allows you to amend a classification or update a variant description, if needed, as you complete your clinical report. To use the Bulk Update feature:

  1. Click the checkbox at the top left box of the Interpret Variants table. This will select all variants
  2. Click the "Bulk Update" button located above the Interpret Variants table.
  3. Under Scoring Status, click the "Set Variants to Scored" check box then click "Apply"

 

Now, you can specify the Report Section for any variant that should be reported. To do this:  

  1. In the Interpret Variants table, Report Section column, there is now a drop-down menu available for all Scored variants. 
  2. Update the report section in which a variant should be reported:
    • Primary Findings
    • Secondary Findings
    • Not Reported (default)

 

Sanger Confirmation

Finally, you can specify whether you want a variant to be confirmed via Sanger sequencing. To do this:

  1. Click the "Show/Hide Columns" button to display the "Confirmation Status" field, if it is not already displayed.
  2. Click the left check box for each variant that you want to be confirmed via Sanger. 
  3. Click the Bulk Update button
  4. Select the "Request Confirmation" radio button, and then click "Apply"

 

You can also manage results from the Sanger confirmation process with the additional statuses in the Confirmation Status column: "Confirmed" and "Failed". See the Confirming Variants via Sanger Sequencing section below for the workflow for Sanger or other external confirmation.

Interpreting Trio and Quad Tests

The data in family reports is similar to panel and exome reports, with the addition of family member data. When you select “Review Results” for a family report, the Interpret Variants page will be displayed with these additional fields available:

  • Mother Zygosity
  • Father Zygosity
  • Sibling Zygosity (for Quad)
  • Mother Quality / GQ / Coverage
  • Father Quality / GQ / Coverage
  • Sibling Quality / GQ / Coverage (for Quad)

By default, the data is ordered by VAAST rank. VAAST is Fabric Genomics' advanced algorithm for ranking disease-causing variants and genes. More information on VAAST is provided in the Advanced Genome Analysis Using VAAST section.

Also by default, Recessive mode of inheritance is displayed. Use the buttons to the left of the table to toggle to another mode of inheritance, or show all modes of inheritance.

Additional candidates based on mode of inheritance beyond those ranked by VAAST are included in the report. With the default sorting based on VAAST rank, these variants will be displayed after the VAAST candidates. The “Source” for these candidates is called “Flex”.

You can also include candidates in the proband that do not fit a standard mode of inheritance. To do this:

  1. Click the “+Add Variants” button above the Interpret Variants table. This will launch Clinical Variant Miner, which which allows you to use rich filtering capabilities to include additional variants for your clinical exome interpretation. For more information on using Variant Miner’s filters, please see the Variant Miner section of the User Guide.
  2. Click on a variant in the Variant Miner table to highlight it in blue.
  3. Click on the “Add Variant to Report” button on the upper right to add the highlighted variants in your interpretation. You should think of this as creating a short list of candidate variants In the Interpret Variants page, you will review this short list of variants in detail and decide on which variants to report.
  4. The “Source” for these candidates is “Manually Added”

For the De Novo mode of inheritance, a Filter False Positives checkbox is available under the Inheritance dropdown menu. This option filters out any variants where both parents do not have a no ref call (both alleles). This is useful when you have Trio and Quad reports generated from vcf files containing Ref Call & No Call data will display open circles for ref calls, and dashes ("-") for no calls for the parents. 

 

Interpreting Duo Tests

Duo reports allow you to analyze a proband and a relative. The report contains data from the proband including a Solo VAAST ranking, and genomic data from the relative.

By default, the data is ordered by VAAST rank. VAAST is Fabric Genomics' advanced algorithm for ranking disease-causing variants and genes. More information on VAAST is provided in the Advanced Genome Analysis Using VAAST section.

Also by default, Recessive mode of inheritance is displayed. Use the Proband's Inheritance (from VAAST) dropdown menu to the left of the table to toggle to another mode of inheritance, or show all modes of inheritance.

You can use the Proband's Genotype and Relative's Genotype dropdown menus on the left, to filter data by genotype and analyze different potential modes of inheritance. 

Additional candidates based on mode of inheritance beyond those ranked by VAAST are included in the report. With the default sorting based on VAAST rank, these variants will be displayed after the VAAST candidates. The “Source” for these candidates is called “Flex”.

The list of candidates in the Duo report includes all protein changing variants. You can also include additional candidates from the proband such as intronic or synonymous variants. To do this:

  1. Click the “+Add Variants” button above the Interpret Variants table. This will launch Clinical Variant Miner, which which allows you to use rich filtering capabilities to include additional variants for your clinical exome interpretation. For more information on using Variant Miner’s filters, please see the Variant Miner section of the User Guide.
  2. Click on a variant in the Variant Miner table to highlight it in blue.
  3. Click on the “Add Variant to Report” button on the upper right to add the highlighted variants in your interpretation. You should think of this as creating a short list of candidate variants In the Interpret Variants page, you will review this short list of variants in detail and decide on which variants to report.
  4. The “Source” for these candidates is “Manually Added”

 

Using Phevor to Leverage Phenotype in your Solo or Family Interpretation

Phevor enables you to use phenotype information to rank disease gene candidates. Phevor directly integrates phenotype and gene function information with genomic data by re-ranking the disease-causing genes from a VAAST analysis using phenotype information. Phevor was co-developed by Mark Yandell’s lab at the University of Utah and Omicia (Singleton et al, 2014).

Phevor can be used in a clinical interpretation where a VAAST analysis has been run, so a Solo, Trio or Quad test.

To run Phevor:

  1. Click the green “Run Phevor” button above the Interpret Variants table
  2. In the Run Phevor modal, enter one or more phenotype terms.You can search on HPO1 terms, synonyms and definitions. You will be prompted with HPO matches as you enter terms.
    • Use specific terms (i.e. dilated cardiomyopathy instead of cardiomyopathy) and try to limit your search to five or fewer terms. Larger numbers of terms or general terms can reduce Phevor’s differential scoring of genes
  3. As you enter terms, details on the most recently entered HPO term will be provided below the search term box. If you click on a more specific sub-node term, it will replace the higher level HPO term.
  4. Run Phevor. Phevor will perform the analysis in real time.

Once the Phevor analysis has completed you will see a new Phevor gene rank column next to the VAAST rank column. You can also mouse over the Phevor rank for more detailed information from the Phevor analysis:

  • Phevor Score: Combined score of VAAST and Phevor Phenotype-Gene association. The Phevor Score is logarithmic, so the separation between scores is important. For instance, 3.0 is ten times larger than 2.0, and 100 times larger than 1.0. Further, Phevor scores less than 1 represent very small values with scores below 0 being insignificant. Several Phevor Scores may group together in a “plateau”. It is often useful to consider all of the candidate genes before the first plateau of scores, then go to the next plateau, etc.
  • Phenotype-Gene Association: score assigned to a gene by Phevor based on strength of association of the phenotype to the gene. The closer to 1, the higher the association.

 

1Sebastian Köhler, Sandra C Doelken, Christopher J. Mungall, Sebastian Bauer, Helen V. Firth, et al. The Human Phenotype Ontology project: linking molecular biology and disease through phenotype data. Nucl. Acids Res. (1 January 2014) 42 (D1): D966-D974 doi:10.1093/nar/gkt1026

 

Confirming Variants via Sanger Sequencing

Variants marked with the status “To Be Confirmed” via Sanger sequencing or a different method can be exported manually with a CSV file or via the Opal API. To export the list of variants, go to the Clinical Reports page and click the Actions drop down menu for your report. Select the “Export Variants to Be Confirmed” option. A CSV file containing the variants to be confirmed will be downloaded to your computer.

To use the Opal API, please contact support@fabricgenomics.com.

 

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