Variant summary

In both the Shortlist tabs and Filter view, each variant is listed with a summary display, grouping the most crucial annotations. To explore more detailed annotations on the variant, the Variant card can be opened.

SNV summary annotations

The following annotations and links are provided in the variant summary display of the SNV/Carrier results and in the filter view:

Gene name (according to HGNC): The menu next to the gene name links to public sources OMIM, PubMed, NCBI genes, HUGO genes, Uniprot and MGI.

Chromosomal location of the variant with links to Ensembl, UCSC, DECIPHER, Mitomap (for mitochondrial variants), Google Scholar, Alamut Visual genome browsers and to IGV. To open variants in either Alamut Visual or IGV, these applications should be opened on your computer and, in case of IGV, the BAM file of the sample of interest should be opened. When clicking the Alamut or IGV links in Moon, these programs will jump to the location of the Moon variant.

rsID of the variant (if available)

ClinVar label if the variant is listed on ClinVar. Clicking the label will open a new tab with the ClinVar page of the variant in question.

Invitae label if the variant is included in Invitae’s internal knowledge base (InvitaeKB).

KB label if the variant is listed on your lab's knowledge base. Clicking the label will redirect you to the KB page of the variant in question, allowing you to review the annotations provided in the KB.

Genotype: The genotype is shown as the combination of Ref and Alt alleles based on the exact genotype call in the VCF (0/1 or 1/1). In the displayed genotype, the reference allele is colored black whereas the alternate allele is colored red. The reference allele is also displayed as such below the genotype. For X-linked calls in males, all variants in the VCF with either 0/1 or 1/1 genotypes, are indicated as hemizygous alternate calls, and considered as such for the analysis.

Predicted effect of the variant (eg. missense, stop gained, frameshift, intronic…).

Protein notation for coding variants (HGVS)

Coding notation: In the overview table, you can switch between protein and coding notation by clicking on it.

Publications: Link to Mastermind (by Genomenon), an extended database of scientific literature. If a specific c. and/or p. notation has ever appeared in scientific literature, the number of publications mentioning the variant in Mastermind is listed. The link redirects the user to Mastermind, where further assessment of the publications can be made.

Disorder name with link to OMIM/PubMed (annotated based on Apollo). For genes linked to more than one disease phenotype, Moon annotates the best disease match based on concordance between the genotype of the patient and the disease’s known inheritance pattern, and phenotypic overlap with the inputted clinical features.

Inheritance pattern of the suggested disease as present in Apollo (eg. AR, AD, XR, XD, MT).

Segregation information in case of a family analysis (de novo, compound heterozygous)

Indication of how many comments have been added to the variant in the current or other cases.

In the Filter view, it is indicated for each variant why the variant was filtered out of the analysis of the automated pipeline. For variants in the shortlist, this field indicates the variant to be shortlisted by Moon.

Warning icons might be shown in the overview table, indicating that some of the annotations are not as expected. Details on these warnings are provided in the variant cards.

Variant card

In both the Shortlist and Filter view, detailed and extended annotations on each variant are provided in a variant card. This variant card can be displayed by clicking the Info icon, which is available at the right hand side of a variant summary display.

Open

The following annotations, grouped by category as in the variant card, are provided for each variant.

General variant info

The top section of the variant card shows the annotated gene name, chromosomal position of the variant, rsID (if available) and labels indicating the clinical classification of the variant in ClinVar, Invitae’s internal KB (Invitae) or the lab's KB (KB).

On the right side of this section, the genotype is displayed as the combination Ref and Alt alleles defined in the VCF. The reference allele is always shown in black and also specifically defined below the genotype presentation, the alternate allele is displayed in red. In the example below, the variant was called in heterozygous state with a C as Ref and an G as Alt, resulting in a 'CG' genotype.

Open

Segregation

This section shows a pedigree for the analysed family configuration. Genotypes of included family members are displayed with reference alleles in black and alternate alleles in red. Affected family members are indicated with a filled symbol in the pedigree. Presence of positive consanguinity is also indicated, as inputted on the Patient info screen.

Literature

In case the c. and or p. notation of a specific variant has been described in scientific literature, it is indicated how many publications are mentioning the variant in question. Clicking this information redirects you to Mastermind (by Genomenon), where the publications mentioning the variant can be further explored.

Disorder

The Disorder section displays the chosen disorder annotation by Moon. Selection of the associated disorder by Moon is based on overlap of the input phenotype with the phenotype of disorders for variants only displayed in the Filter view, and based on the concordance between the variant's genotype and known inheritance patterns for the disorder for variants displayed in the Shortlist after automated analysis.

The inheritance pattern of the annotated disorder is indicated, in addition to the known age of onset range for the disorder. A warning sign is shown in case the known age of onset range of the disorder significantly differs from the age of onset of symptoms of the patient. In the Carrier tab, if the SNV is in compound heterozygosity with a SV/CNV, this is indicated here too.

Below the disorder indication, a HPO checklist is displayed indicating which of the input HPO terms have been associated with the disease suggested by Moon. Moon highlights both direct and indirect matches with the input HPO terms (through relations up to 2 levels up and down in the HPO ontology). Hovering over the checkmark icon brings up a tooltip that indicates in which order the HPO term matches with terms known to be associated with the annotated disorder.

Whether the gene is known to be subjected to imprinting is also indicated.

Effect

Variant effect annotations in all available transcripts, protein and coding notations and exon ranks are indicated. Clicking the transcript IDs opens the overview page of the transcript in the respective database. Clicking the c. notations redirects you to the specific patient’s mutation page in Alamut Visual for that specific transcript.

Variant effect annotations in Moon (c. notation, exon rank etc.) are based on the associated Ensembl ENST as reference. The RefSeq NM IDs shown in the table are a translation of the ENST transcripts. For the majority of transcripts, there is a unique mapping of ENST to NM. For ENST transcripts that map to multiple NM IDs, Moon displays the MANE transcript where available. For the remaining ENSTs without unique mapping to a RefSeq NM ID, Moon only displays one of the alternative NM IDs. Due to this, it is possible that the NM ID shown in Moon is not in line with the c. and p. notation annotated based on ENST, and shown on the same line.

Protein prediction

Conservation scores and protein prediction scores are displayed as coloured dots. Details on the specific values for each score can be displayed by clicking in the Protein prediction section of the card.

Scores are highlighted in red if significant as compared to a threshold set in Moon. The threshold in Moon was set based on recommended thresholds in scientific literature. The following thresholds are used as cutoff for highlighting them in the variant card: SIFT <= 0.05; Polyphen > 0.85; MutationTaster > 0.31713; MutationAssessor > 3.5; FATHMM <= -1.5; VEST3 > 0.85; CADD > 15; DANN > 0.85; FATHMM MLK > 0.8; Eigen Phred > 15; GERP > 2; Phylo P > 0; Phastcons > 0.5; SiPhy > 3; EVE > 0.762.

Splice prediction

Two sets of splice predictions scores are annotated in Moon and displayed in the variant cards:

• Splicing ADA and Splicing RF scores are annotated for variants at positions −3 to +8 at the 5’ splice site and −12 to +2 at the 3’ splice site. These are considered significant if > 0.60. More info on these splice site prediction scores is available in the publication by Jian et al. (2014; PMID 25416802).

• SpliceAI: the probabilities of a variant affecting splicing within a 50 nt window around it, divided in probabilities of Acceptor/Donor Loss/Gain. Scores higher or equal to 0.2 are considered as significant in the automated analysis, and in the UI (coloured in red in the variant card).

A link is also provided to the SpliceAI lookup tool from the Broad Institute to allow checking SpliceAI scores for variants for which no SpliceAI annotation is currently available in Moon (eg. deep intronic variants).

Gene region

This section shows a rough gene visualisation indicating the intron-exon structure of the gene according to the indicated transcript and the position of the detected variant(s) (indicated with an arrow on top of the visualisation).

Clicking the "Expand" icon opens the Moon SNV genome browser at the variant's position. This allows for easy exploration of the surrounding region of the variant from within the variant card.

Frequency

This section shows the gnomAD allele frequency of the variant, with indication of the number of homozygotes, heterozygotes and hemizygotes in this database for the corresponding sample type (WES/WGS).

The gnomAD frequency is clickable and redirects users to the gnomAD webpage of the variant of interest, where eg. population specific frequencies can be reviewed.

The Lab frequency represents the allele frequency of the variant based on all samples uploaded in the Moon lab account. The number of homo/hemi/heterozygous observations is also indicated. Clicking either the frequency value or observations, redirects you to the case repository page where details on these samples can be explored.

The Diploid frequency represents the allele frequency of the variant based on all currently present samples on the used server, and also includes samples that seeded this database.

Quality

Variant quality parameters are extracted from the VCF file and displayed. These include read depth at the position of interest (DP), allele depth (AD) and genotype quality (GQ). A warning is shown in case of reduced variant quality. A low quality annotation can be a result of low overall DP, low AD for one of the alleles, allelic imbalance, low GQ, or the site being considered multiallelic by Moon (5 or more Alt alleles present in samples uploaded on the server).

Comments

At the bottom of the variant card, a text box is provided to add a comment to the variant. Double clicking your text allows text formatting and adding links. Details on who added the comment and when, are added upon saving. The comment can be easily removed by the person who added it, by clicking the trash icon.

Comments are visible to all members in the lab account. Comments will also be shown in other samples if the same variant is present, with reference to the sample in which the Comment was originally added.