Alamut™ Visual Plus FAQs

gnomAD data are displayed in the Allele Frequency Databases track. Right-clicking on the variant of interest reveals the variant panel with details from gnomAD shown in a box within Alamut^TM Visual Plus. At the top of the gnomAD box you will see Genome and Exome tabs, which, if selected, are highlighted in blue and information is shown in the box. Clicking on the hyperlinked “gnomAD (vx.x.x)” tab will take you to the gnomAD page for the variant. Note that depending on the type of data and which filters are used, the text will be different, but it will start with gnomAD.

By default, Alamut^TM Visual Plus includes all available variants from GnomAD, irrespective of the filters or the filter cut-off.

There are three filters – the “PASS”, RF, and AC0 filters. Each of these filters has cut-offs defined by gnomAD, for both Exome and Genome data. If you only want to view variants that have passed through all the quality filters, tick the box “PASS only” on the gnomAD track for Alamut^TM Visual Plus, deciding if you want to see Exome or Genome data, or both.

• AlamutTM Visual Plus currently uses Genome Aggregation Database Version 2.1 for GRCh37/hg19 genome build. The link to this version of gnomAD is:  https://gnomad.broadinstitute.org/variant/1-55516888-G-GA?dataset=gnomad_r2_1 . 
• For GRCh38/hg38 genome build, gnomAD 4.0.0 is now available in AlamutTM Visual Plus. The link to this version of gnomAD is:  https://gnomad.broadinstitute.org/variant/1-55051215-G-GA?dataset=gnomad_r4

With 730,947 exomes and 76,215 genomes, the gnomAD v4.1 call set is the largest of all the gnomAD versions. It also contains nearly all data from prior versions (v2 and v3) except a small number of samples excluded due to data quality and updated sample filtering pipelines. Since gnomAD v4.1 is mapped to GRCh38, if you haven’t switched to GRCh38, now is the time!

gnomAD links are built based on the position, ref, and alt of the variant. In AlamutTM Visual Plus, if we apply the 3′ rule, those values may change, which directly affects the url functionality.

We compute Genotype Count based on values from a gnomAD VCF. In some cases, we end up with negative counts due to discrepancies in the initial values provided in the VCF. This is the way gnomAD is handling these variants. 

RefSeq displays GRCh38 as a default genome for all rsID links. On dbSNP variant pages, links for the GRCh37 genome build are usually available.

This is due to occasional genome/transcript sequence discrepancies, where the genome reference includes polymorphism minor alleles, but the transcript includes corresponding major alleles. This means that some genomic variants are seen as ‘non-variants’ if analyzed at the transcript level.

Basically, at the positions highlighted in red, the nucleotide of the transcript differs from the nucleotide of the genome build (GRCh37 or GRCh38). For these nucleotides, it is more difficult to definitively determine whether a variant is indeed a variant.

These discrepancies mainly occur in RefSeq transcripts (Beginning with “NM_”), as RefSeq does not correct the transcript to the genome build, while ENSEMBL transcripts (beginning with “ENST”) are corrected to match the nucleotides present in the genome build.

Two different conventions are used for exon naming.
Systematic Exon Numbering starts at 1 and counts and numbers each exon numerically. So, if there are 10 exons, for example, they will be numbered from 1 to 10.
Whereas Custom Exon Numbering is the historical numbering that was determined when the gene was first sequenced. The Custom Exon Numbering originally included splicing variants. For example, if there was a splicing difference, you could have an exon numbered 10a in one transcript and 10b in another transcript. With Systemic Exon Numbering, this exon would just be numbered 10 if it was the 10th exon. The Custom Exon Numbering usually comes from the original paper and/or the scientist that determined the sequence. This had previously been supported by NCBI but was discontinued several years ago in favor of Systematic Exon Numbering. However, researchers still use Custom Exon Numbering for genes such as BRCA1 and BRCA2.
In the AlamutTM Visual Plus toolbar, if you click on “Exon Naming” you can change between the two naming conventions. Alternatively, you can set the program to “use systematic exon numbering by default”. To do this, open the “Alamut Visual Plus” menu > “Preferences”, and then select this setting in the “View” tab.

Two different conventions are used for exon naming.
Systematic Exon Numbering starts at 1 and counts and numbers each exon numerically. So, if there are 10 exons, for example, they will be numbered from 1 to 10.
Whereas Custom Exon Numbering is the historical numbering that was determined when the gene was first sequenced. The Custom Exon Numbering originally included splicing variants. For example, if there was a splicing difference, you could have an exon numbered 10a in one transcript and 10b in another transcript. With Systemic Exon Numbering, this exon would just be numbered 10 if it was the 10th exon. The Custom Exon Numbering usually comes from the original paper and/or the scientist that determined the sequence. This had previously been supported by NCBI but was discontinued several years ago in favor of Systematic Exon Numbering. However, researchers still use Custom Exon Numbering for genes such as BRCA1 and BRCA2.
In the AlamutTM Visual Plus toolbar, if you click on “Exon Naming” you can change between the two naming conventions. Alternatively, you can set the program to “use systematic exon numbering by default”. To do this, open the “Alamut Visual Plus” menu > “Preferences”, and then select this setting in the “View” tab.

Ensembl and RefSeq transcripts differ in that Ensembl transcripts are mapped onto the reference genome, whereas RefSeq transcripts are mapped onto mRNA sequences. Due to differences between reference genomes and individual mRNAs, some RefSeq mRNA’s might not map perfectly to the reference genome, resulting in the possibility of small differences between Ensembl and RefSeq transcripts. AlamutTM Visual Plus uses Splign (a tool developed by RefSeq) to align all transcripts to the genome build.
For more information please see: https://www.ensembl.org/Help/Faq?id=294

Alamut^TM Visual Plus includes a splicing module accessible from the variant panel that integrates a number of prediction algorithms. It provides the user with automatically-computed prediction scores.
A brief description of splicing signal prediction can be found on page 63 available here: https://extranet.interactive-biosoftware.com/User%20Guide%20Alamut%20Visual%20Plus%20v1.6.1.pdf

Alamut^TM Visual Plus computes splicing scores based on implemented algorithms. The user is responsible for interpreting these scores based on the scientific context and peer-reviewed guidelines. 
We can suggest the following paper to help you interpret splicing scores: https://pubmed.ncbi.nlm.nih.gov/22505045/

And the followingvideo from ClinGen: https://clinicalgenome.org/tools/educational-resources/materials/splicing-and-in-silico-splicing-predictors/

The user manual section on splicing (from pg 63) provides an overview of this topic, as well as links to publications and more splicing-related information: https://extranet.interactive-biosoftware.com/User%20Guide%20Alamut%20Visual%20Plus%20v1.6.1.pdf

The following paper also provides a good overview of this topic: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC275472/
The first sentence of the abstract states: “Cryptic splice sites are used only when use of a natural splice site is disrupted by mutation.”

Overall, the literature suggests that both natural and cryptic splice sites are important, but their relevance depends on the context of your investigation.

SHANK3 is quite a problematic gene. Alamut^TM Visual Plus mapped the available transcripts to genome builds and NM_033517.1 could not be mapped to either genome build, due to mismatches between the transcript and builds.

We do, however, have a RefSeqGene NG_008607.2, which is based on the transcript NM_033517.1

SIFT scores are calculated based on multiple sequence alignments of protein orthologues (SIFT Aligned Sequences). Scores differ between builds 37 and 38, because MSA (Multiple sequence Alignments) can be different depending on the genome used. MSA are accessible by clicking on the missense prediction tool button in the Variant Panel. PolyPhen-2 (in AlamutTM Visual Plus) does not use MSA, just the human protein sequence and the substitution information.

SIFT 6.2.0
Polyphen2

The following page explains possible reasons for inconsistencies between versions: http://genetics.bwh.harvard.edu/pph2/dokuwiki/faq. Our team is continuously working to harmonize and update the protein sequence database used to build the multiple sequence alignment by PolyPhen-2. This could explain the differences in the scores.

The Polyphen-2 prediction scores automatically displayed in AlamutTM Visual Plus are extracted from the WHESS database (http://genetics.bwh.harvard.edu/pph2/dbsearch.shtml). The WHESS database contains a pre-computed set of PolyPhen-2 predictions for the Whole Human Exome Sequence Space. 
Whereas, the scores obtained on the site using the batch query (http://genetics.bwh.harvard.edu/pph2/bgi.shtml) are generated upon each request. 

SIFT scores are calculated based on multiple sequence alignments of protein orthologues (SIFT Aligned Sequences). The scores can differ between genome builds 37 and 38 because MSA (Multiple sequence Alignments) can differ depending on the genome used. Differences seen between the Alamut^TM Visual Plus in-house predictors and the predictor website can be because the MSA differs and/or because the algorithm version differs between the website and the in-house versions.

In Alamut Visual and AlamutTM Visual Plus, the SIFT missense predictors are computed using the orthologues alignment. Differences in SIFT scores can be explained by differences in orthologue alignment. Alamut Visual contains ‘in-house’ orthologues for some genes, whereas AlamutTM Visual Plus contains Ensembl orthologues.

The Orthologue Alignments for each gene are downloaded from Ensembl Compara (https://www.ensembl.org/info/genome/compara/index.html). Differences between GRCh37 and GRCh38 are due to the species used in the alignments not being the same in all cases. The data for GRCh38 are more up to date in Ensembl, but it is extremely difficult to determine which alignment is better. AlamutTM Visual Plus includes a standard set of species, but this is dependent on what is available in Ensembl Compara. These sequence alignments are used for missense predictions in AlamutTM Visual Plus.

See answer 21.

See answer 21.

Alamut Visual uses conservation scores from UCSC: https://genome.ucsc.edu/cgi-bin/hgTrackUi?db=hg19&g=cons46way, whereas, AlamutTM Visual Plus uses a more up-to-date set of conservation scores.

• Alamut Visual
  • PhastCons
    • GRCh37 – 46 vertebrates (2013)
    • GRCh38 – 100 vertebrates (2013)
• AlamutTM Visual Plus
  • PhastCons
    • GRCh37 – 100 vertebrates (2018)
    • GRCh38 – 100 vertebrates (2018)

Catalogs are updated at the same frequency as the release of those catalogs. For example, Clinvar is updated bi-monthly, whereas catalogs such as dbSNP are updated yearly, and other catalogs less frequently. GnomAD is updated when a release is available that will cover all AlamutTM Visual Plus requirements.

In AlamutTM Visual Plus, the limit fixed to trigger NMD is 50 nucleotides.

Users do not have direct access to the AlamutTM Visual Plus database and thus do not need to write SQL queries to access the data. All relevant data are available through the AlamutTM Visual Plus interface by clicking on different tabs.

This is related to mismatches that can exist between the transcript and reference genome. Alamut Visual only uses transcript data, while AlamutTM Visual Plus displays both transcript and reference genome sequences.

Several RefSeq transcript versions cannot be added to the Alamut^TM Visual Plus database, due to significant mismatches with the reference genomes GRCh37 and GRCh38. Alamut^TM Visual Plus uses Splign (https://www.ncbi.nlm.nih.gov/sutils/splign/splign.cgi), a RefSeq alignment algorithm, to ensure that transcripts align successfully with the reference genomes before adding them into the Alamut^TM Visual Plus reference database.

It is possible to click on the “Alamut” button in the SOPHiA DDM^TM Platform, or to download BAM files from WES (or from targeted panels or even from whole-genome sequencing). In Alamut^TM Visual Plus, the BAM file will be loaded by segment and visualized by gene due to the index file (.bai) associated with the BAM file. The BAM file will not be entirely loaded for all genes at once, meaning that the BAM file can be of any size. 

The nucleotide conservation track shows scores of evolutionarily conserved nucleotides based on phylogenetic studies between species. Nucleotide conservation scores are extracted from PhastCons statistical algorithms, represented by grey color. The red color means that the indicated value of one nucleotide is higher than what the height of the stick symbolizes. The threshold is fixed at 4 (as for the UCSC). These colors are visible when viewing values in the tooltip.

It is not possible to have more than one transcript related to a single variant in the same database. A new database will need to be created for a different transcript.

Alamut^TM Visual Plus applies the internationally recognized HGVS nomenclature. In the case of reverse genes, the 3’ rule is applied. For all descriptions, the most likely 3’ position of the reference sequence is arbitrarily assigned to have been changed. The 3’ rule also applies to changes in single residue stretches and tandem repeats (nucleotide or amino acid). The 3’ rule applies to ALL descriptions (genome, gene, transcript, and protein) of a given variant. See: http://varnomen.hgvs.org/recommendations/general/

When creating a new variant in an intergenic area, Alamut^TM Visual Plus looks for the two closest genes (upstream and downstream). If no gene is found in a 10,000,000-nucleotide area around the variant position, the “No nearby genes are available” message is displayed.

The export of external annotation is allowed per variant from the Variant Panel or directly from the ‘Variant Exporter’ window.

It is not recommended to install Alamut^TM Visual Plus on a shared drive because of performance issues and potential application instability. The best way to install Alamut^TM Visual Plus is:

• Run Alamut^TM Visual Plus executable (.exe) locally and do not store it on a shared drive.
• When installing the application, select a local settings folder.
• To share variant databases, one of your users can create a new Local Variant Database from the menu and choose to store it on a shared drive. This database can later be imported by any user that has access to this location.
  • If a database is stored on a shared drive or disk, it should be flagged as “Shared Database”.

If the sequencer used to generate the ab1 files is Applied Biosystem 3130XL / ABI3130XL:

To load a Sanger file, Alamut^TM Visual Plus uses the “PBAS” tag of the ABIF format. The “PBAS” tag contains the base-called sequence (i.e the nucleic sequence identified from the electropherogram).

Applied Biosystem 3130XL / ABI3130XL sequencer does not do the base calling step. In that case, sequence analysis software has to be used to do the base calling and to generate a Sanger file compatible with Alamut^TM Visual Plus.

See here for more info about the ABIF format: https://projects.nfstc.org/workshops/resources/articles/ABIF_File_Format.pdf