The Settings dialog is used to customize and modify workflow designs for variant analysis. This dialog offers a range of capabilities, which are discussed below.
Enrichment kits are used in the pre-sequencing DNA preparation step where DNA sequences are directly amplified or captured. With enrichment kits, only part of the whole-genome is enriched and sequenced, without sequencing the entire genome of the sample. This is used in the secondary analysis step. By default, 13 enrichment kits are provided and each can be edited using the edit icon.
Selecting New in the upper right of the Enrichment kit interface, allows enrichment kits to be added to the selectable options. The following details need to be entered for new enrichment kits:
- NAME: Name of the enrichment kit
- DESCRIPTION: Details of the enrichment kit
- Filtering: Defining what variants will be captured when applied to the samples. This includes:
- Include ref/ref: homozygous reference variants will be captured when enrichment kit is applied
- Include all modifiers: captures genetic variants that can modify the phenotypic outcome of the primary disease-causing variant
- Merge BED with ClinVAR BED: This utilizes the existing BED file and captures variants that are also present in ClinVar.
Once a new enrichment kit has been added, click on the hyperlink from the Enrichment kit interface. In the top right of the following window, select the Add icon.
This will provide the ability to add the BED file to the enrichment kit. The available fields include:
- TYPE: This includes Sequencing and Genotype. Genotype is used for a BED file that contains individual genetic variant locations. Sequencing is used for BED files that encompass regions of DNA to capture.
- GENOME BUILD: hg19/GRCh37 or hg38
- BED FILE: This includes ability to Select Existing, Select Shared, and Upload a new file. To upload a new file, select the Browse icon to locate the BED file. Once selected, click Save, and it will convert the BED file into a new enrichment kit that can be used.
For analysis that target a select subset of genes, the gene list can be integrated into the workflow using the Gene Panels option. To create a new gene list, select the New icon in the top right corner. To edit a gene list, select the edit icon next to each panel. All genes must be entered according to the HUGO Gene Nomenclature Committee (HGNC). If a gene is not recognized correctly, there will be a notification for the gene that was incorrect.
The options available for a gene panel include:
- NAME: Name of the gene panel
- DESCRIPTION: Additional details about the panel
- CATEGORY: What category the panel is defined as
- SYMBOLS: List of gene names
- CREATED BY: What user created the gene panel
- CREATED: When was the panel created
- MODIFIED BY: If applicable, what user made modifications to the gene list
- MODIFIED: If applicable, when modifications were made to the list
Variant maps are target capture regions defined by a BED file. Selecting the New icon, will provide a dialog to enter the following:
- NAME: Name of the variant map
- DESCRIPTION: Details of the variant map
- LOCATIONS: Enter the locations in a BED file format. You can also paste from a BED file
The Sequencing Machine refers to the instrument that was used during the primary analysis. If the sequencing machine is not available by default, selecting the New icon will allow the creation of one.
Data Sources refers to the location of the fastq files that can be used in the secondary pipeline for variant calling. The storage devices include:
- Amazon S3: Amazon Simple Storage Service is offered by Amazon Web Services that provides object storage through a web service. This uses the same scalable storage infrastructure that Amazon.com uses to run its global e-commerce network.
- FTP: File Transfer Protocol is a standard communication protocol used for the transfer of computer files from a server to a client on a computer network. FTP is built on a client-server model architecture using separate control and data connections between the client and the server.
- BaseSpace: A powerful website where biologists and informaticians can easily store, analyze, and share genetic data.
Case Sub Statuses refers to cases that have been assigned to an individual user.
The ACMG Settings is used to modify specific thresholds that are used in the ACMG criteria for variant analysis. This includes:
- REVEL PATHOGENIC CUTOFF: In general, a score > 0.75 is considered evidence of pathogenicity (ClinGen Variant Curation SOP)
- REVEL BENIGN CUTOFF: Suggested REVEL (Rare Exome Variant Ensemble Leaner) cutoff
- SIFT CUTOFF: Positions with normalized probabilities less than 0.05 are predicted to be deleterious, those greater than or equal to 0.05 are predicted to be tolerated.
- PHYLOP CUTOFF: PhyloP scores are also used to remove sites where mutations may be affected by purifying selection. It is usually assumed that a PhyloP score cutoff of < 0 effectively filters out sites under selection.
- GERP CUTOFF: GERP scores are also used to remove sites where mutations may be affected by purifying selection. It is usually assumed that a GERP score cutoff of < 2 effectively filters out sites under selection.
- DBSCSNV CUTOFF: Threshold value for dichotomous effects suggested by dbscSNV
- METASVM CUTOFF: Threshold cutoff value suggested by MetaSVM
- BP4/PP3- ALLOW BOTH TO BE MARKED: In case of contradictory evidence (both BP4 and PP3), if checked, both will be marked. If not checked, neither will be marked.
The Protocols dialog will display workflows that can be utilized for variant analysis. The protocol defines the filtering logic and genetic models that are used to identify the clinically relevant variants in a streamlined fashion. Protocols can be used for standardization of workflows as it defines the templates, genetic models, report, allele frequency thresholds, and phenotype for the variant analysis. This includes:
- NAME: Name of the protocol
- TYPE: Category of the protocol
- DESCRIPTION: Details of the protocol
- PHENOTYPES: Associated phenotypes
- DISEASE: Associated diseases
- TEMPLATE: Name of the template
- CREATED BY: Lists the user who created the protocol
- CREATED: When the protocol was created
- MODIFIED BY: Lists the user who made modifications to template
- MODIFIED: Lists when the modifications were made.
To create a new protocol, select the New icon in the top right of the protocol interface. This will provide the following options to create the protocol, which can then be used for all downstream analyses. Fields includes:
- NAME: Name of the protocol
- TYPE: Focus of the protocol, includes:
- Health Screening
- Panel Design
- ORDINAL: Position in series
- SERIAL NUMBER: Used for protocol identification
- AUTOMATIC ANALYSIS: This applies pre-defined filter logic to the analysis and the remaining variants will automatically be incorporated into a clinical report.
- AUTO ANNOTATE: If a variant is reported on in one case and is seen in another, this feature will apply captures selection and implement the same relevance, classification, and comments.
- DESCRIPTION: User applied details of the protocol
- PHENOTYPES: Associated phenotypic terms, if applicable, can be entered
- GENETIC MODELS: Different logic used for variant filtering can be applied, options include:
- Fast Track
- Recessive Hom
- Recessive Compound HET
- Dominant HET
- HET- Cancer
- HOM- Cancer
- Other Findings
- Incidental Findings
- ALWAYS SHOW PAT/LP: Enables filtering logic to always display pathogenic and likely pathogenic variants according to ClinVar.
- DEFAULT GENETIC MODEL: The default genetic model is the model that is displayed first when opening the variant analysis interface.
- DEFAULT ENRICHMENT KIT: Enrichment kits are used in the pre-sequencing DNA preparation step where DNA sequences are directly amplified or captured. If there is a custom enrichment kit, it can optionally be selected.
- DISEASE : The disease related to the protocol
- DISEASE FREQUENCY (%): This figure should represent the expected percentage of individuals presenting the symptoms in the general population and NOT the allele frequency. The default is 0.1% which means that the disorder is found in less than 1 / 1000 individuals.
- CALCULATE LOCAL FREQUENCY: This option will calculate the local frequency of the variants across internal samples.
- PROBAND SAMPLE SOURCE: Defines the location the DNA was collected. This includes:
- Tumor Biopsy
- REPORT TEMPLATE: Defines the report template that will be generated following variant analysis and evaluation. This includes:
- Carrier Screening
- Health Screening
- Rare Genetics CNV
- Rare Genetics Default
- Tumor Default
- DEFAULT SORT BY COLUMN: Provides capability to sort variants in the variant analysis interface by an annotation field.
- SORT DIRECTION: Option to sort default column by ascending or descending.
- EXPAND COLUMNS: Defines which annotation sources are expanded in the variant analysis by default. This includes:
- Matched phenotypes
Once the fields have been assigned for the new protocol, select the Save icon. This new protocol will be stored with the Geneyx Analysis account and can be used for all downstream variant analysis and for repeated workflows.
The Filters dialog allows the modification of logic that is used in each protocol. Available fields include:
- NAME: Name of the filter logic
- DESCRIPTION: Details of the filter logic
- CATEGORY: Category of the filter workflow
- GENETIC MODELS: Which genetic model the filter is applied to
- QUERY: a Boolean JS expression that is calculated on every VCF data row – it should return true or false
- CREATED BY: What user created the filter
- CREATED: Date filter was created
- MODIFIED BY: What user made modifications to the filter
- MODIFIED: Date modification of filter was made
Default filters include:
- ACMG: Filter to all genes within the ACMG list
- CompHet genes: Includes only genes with 2 or more candidate variants
- CompHetTrio: Compound Heterozygous for trio workflows. Two distinct heterozygous variants in the same gene, where one is derived from only the mother and the other is derived from only the father.
- FastTrack-AF: Not prevalent in your lab samples
- FasTrack-Classification: Not benign according to ACMG and ClinVar
- FastTrack-Clinical: OMIM & segregation. Must be likely pathogenic in ClinVar and ACMG, present in OMIM gene with disease, homozygous for recessive disorders, and if a trio workflow, must be compound heterozygous.
- FastTrack-Impact: Removes low severity variants that don’t have info from ClinVar and filters out inframe indels with repeat region without domain
- FastTrack-Quality: Quality based on % ALT (homozygous/hemizygous with >80% ALT reads, and heterozygous variants with 20-80% ALT reads)
- Hetero variants: Includes heterozygous variants only
- Homo variants: Includes homozygous variants only
- Max AF Dom: Filter according to the maximal available allele frequency for dominant genetic model
- Max AF Rec: Filter according to the maximal available allele frequency for recessive genetic model
- Med-High Effect: Exclude variants with the lowest predicted effect (Silent, Start gained, remote splice site position, not damaging by all prediction tools
- Med-High Quality: Exclude low reliability variants with coverage less than 10X and quality score (GQ) less than 15
- Mitochondria: Mitochondria mutations only
- No Mitochondria: All mutations beside mitochondria
- OMIM Genes: Filter for OMIM genes
- SV-AF: Structural variation allele frequency
- SV-Effect: Structural variation effect
The Secondary Pipelines lists the available options for variant calling. This includes:
- DRAGEN Exome: Enables ultra-rapid analysis of NGS data for whole exomes
- DRAGEN Exome (Clinical Exome): Enables ultra-rapid analysis of NGS data for a limited number of genes
- DRAGEN Exome (Panel): Enables ultra-rapid analysis of NGS data for a targeted gene panel
- DRAGEN Exome (Target Region): Enables ultra-rapid analysis of NGS data for targeted regions
- DRAGEN Whole Genome: Enables ultra-rapid analysis of NGS data for whole genomes
The Account Management provides the ability to grant permission, roles and access to specific users within an organization.