Regul@tionSpotter Documentation

Analysing a VCF file

Input

VCF file

Analysis settings

Homozygous Only

Tick this box if you want to only consider homozygous variants in your analysis.

Filter polymorphisms

Enter a threshold for discarding variants as polymorphisms using the frequency observed in 1000G and ExAC. The default filtering is 4 for homozygous in 1000G and 10 for homozygous in ExAC. It is also possible to filter for variants present in any form (heterozygous and homozygous) as defined in the second row in case of non-recessive traits. Set both values to zero if you do not wish any filtering. All values refer to the number of individuals with the specific allele setup.

Minimum Coverage

Enter the minimum value for your variant's coverage. The default is 4.

Analyse the following regions or genes

Select whether you wish to analyse the entire VCF file, or custom regions or genes. This can also be done after analysing the complete vcf file upon restricting the display of analysis results to variants residing in certain candidate genes or regions. There is no difference between the two filter options regarding the output. However, speed of the analysis is usually increased if the restriction to certain genes / regions is already selected ahead of the analysis. On the other hand, users are free to select / deselect different gene panels after the analysis if they initially do not exclude any variants.

Synopsis and display settings: RegulationSpotter landing page

A page similiar to this page will appear after RegulationSpotter is done with its analysis. You can navigate to a synopsis of your analysis here as well as filter and sort your results.

Project ID

The project ID allocated by RegulationSpotter. It is used in the URL to enter your project results. If you wish to easily access your results later on, you can note the URL and the project ID. Please do not delete or alter the project ID in the text field on the results page.

Synopsis

Submitted Variants

Number of alterations (lines) in VCF file.

Discarded before analysis

Number of variants which were filtered out according to user input (below coverage, not homozygous, out of specified region / chromosome) or due to input / format errors (e.g. variant equals refseq, reference allele equals alternative allele, Indel is too long or neither genotype nor frequency are supplied).

intratranscriptic variants (MT +RS)

Variants localised in a protein-coding transcript, which were analysed with MutationTaster for evaluation of effects on the protein, as well as by RegulationSpotter, to account for possible regulatory impact of intratranscriptic variants.

MutationTaster analyses (variant/transcript pairs)

The lines following this subheading give information about different types of variants and predictions made by MutationTaster. The sum of all predictions made by MutationTaster are usually significantly more than the analysable variants, because for most variants more than one (suitable) transcript will be found.

Type

The type of prediction algorithm used by MutationTaster. without_aae: our algorithm for silent alterations. simple_aae: our algorithm for simple amino acid exchanges (one amino acid exchanged by another). complex_aae: the algorithm for amino acid exchanges involving more than a simple exchange of one residue.

Prediction

Gives an overview of the predictions generated by MutationTaster. The four options are:

• disease_causing automatic: known to be disease-causing from databases (currently ClinVar).
• disease_causing: probably deleterious. MutationTaster classified the alteration as disease-causing.
• polymorphism: probably harmless. MutationTaster classified the alteration as a polymorphism.
• polymorphism_automatic: known to be harmless from databases (currently 1000G and ExAC).

More information on the classifications can be found in MutationTaster's documentation.

Regulation Spotter only / extratranscriptic variant (RS only)

Number of variants which are located outside of protein-coding transcripts and analysed exclusively by RegulationSpotter. Please note: The sum of intratranscriptic variants plus extratranscriptic variants is not necessarily identical to the number of all variants minus the discarded alleles because one line in the vcf file can contain more than one alternative allele which are all individually counted if discarded.

Filter and display options

Phenotype

You can enter diseases or symoptoms relevant for the case that you are analysing. As soon as you are typing, autocompletion will suggest relevant terms / entries from the HPO, OMIM and Orphanet. Tick the diseases and symptoms of your choice. Then hit the "find suitable genes" button and RegulationSpotter will automatically suggest candidate genes which are known to be associated with the selected diseases or symptoms. A cut-off for gene search can be adjusted. The default is 70%; lowering this value will result in the suggestion of more candidate genes (and more noise), increasing this value will lead to the suggestion of fewer, but highly relevant candidate genes.

Candidate genes

Candidate regions

Variant selection

Choose if you want to display all analyses made by RegulationSpotter and MutationTaster, or only a selection. The default setting is the display of analysis results from both tools.

• 'show intragenic vars' - remove the tick if you do not want to inspect MutationTaster analysis results for variants located within protein-coding genes.
• 'show extratranscriptic vars' - remove the tick if you do not want to inspect RegulationSpotter analysis results.

Hide unlikely variants

Choose if you want to prevent certain variants from being displayed, beause they are at least in the first place rather unlikely to be involved in the onset of disease.

• predicted polymorphisms: variants which are predicted to be harmless (can be either a prediction by MutationTaster or due to presence of this variant in databases such as 1000G and ExAC)
• silent intragenic variants: variants located within protein-coding genes but without direct effect on the amino acid sequence. Some of these 'silent' variants can nevertheless be disease-relevant, however, in a first, rigid filter strategy they are usually left out.
• prediction problems: any kind of MutationTaster prediction problems. If you have already thoroughly inspected all analysis results without finding promising variants, it might be worth it to inspect the variants with prediction problems.

Sort by

This section allows you to sort your results by various properties. By default, they are displayed by effect first, followed by position and effect.

Project management

• export this project: export all analysis results in tab-separatet flat file format. The zip-archive contains a readme file explaining the different columns in the export file. Filter options do not work for export. We plan to offer additional export functions and formats in the near future.
• keep this project: click here if you want your project to be kept available longer than the default of 3 weeks. Once you have chosen this option, deletion via the adjacent link is not possible anymore. If you want to delete your formerly marked as "keep" project, please drop us an E-Mail and we will delete your data on demand.
• delete this project: click here if you want to delete your project. If you do not choose to delete your project, it will nevertheless automatically be deleted after 3 weeks.

Output: Overview

Results table - data

Chr, Pos, Ref, Alt

Information on the location and nature of the alteration.

(known) variant effect

Known or likely effect of the alteration. Depending on whether the variant is considered to be intragenic or extragenic (extratranscriptic), the options are:

Intragenic variants analysed by MutationTaster

• disease causing (ClinVar): known disease mutation listed in ClinVar.
• disease causing: predicted by MutationTaster as disease causing.
• polymorphism: predicted by MutationTaster as harmless.
• polymorphism automatic: known to be harmless from databases.

More information on the classifications can be found in MutationTaster's documentation.

Extratranscriptic variants analysed by RegulationSpotter

• disease causing (ClinVar): known disease mutation listed in ClinVar.
• functional region (much evidence): Regulatory information is available for the variant's location in several data sources, resulting in a high Region Score which makes it principally possible to classify this variant as "functional" with high confidence. The confidence was drawn from testing the distribution of Region Scores for a set of known extratranscriptic disease variants and known harmless extratranscriptic variants with a high frequency of homozygous occurence in the data of 1000G. Due to the available data, RegulationSpotter considers it vary likely for the variant to have a regulatory function. The high confidence label is given to variants for which the positive predictive value (PPV) was at least 98% and the negative predictive value (NPV) was below 98%.
• functional region: Regulatory information is available for the variant's location which leads to a high Region Score. Due to the available data, RegulationSpotter considers it possible for the variant to have a regulatory function.
• non-functional region: Low Region Score which makes it rather unlikely that this variant is located in a regulatory region and plays a functional role. Due to the available data, RegulationSpotter considers the variant to be not located in a regulatory region. For the high confidence label, the same thresholds as for functional variants were used.
• non-functional region (much evidence): Very low Region Score which makes it very unlikely that this variant is located in a regulatory region and thus this variant is very likely not functional in terms of gene regulation.
• polymorphism: The variant was found in homozygous state in several samples in the data from 1000G or ExAC which is indicative of a harmless polymorphism.

Variant Frequency

Information on the availability of the variant in genetic frequency databases (dbSNP, 1000G)

Results table - colour-coded matrix

Most Severe Result

Most severe RegulationSpotter result for all available transcripts, will be used for sorting.

Region Score

RegulationSpotter score for the variant's location. The score integrates all found evidence for the functionality of the variant. Higher values indicate a higher probability of functionality.

Type

Type of alteration: Single nucleotide variant (SNV), Insertion/Deletion (InDel). InDels can be long (>10 bp) or short.

Intragenic Variant

Indicates whether the variant is located within a gene.

NMD / PTC / frameshift /truncated

Indicates whether the variant is a highly deleterious one, e.g. leading to nonsense-mediated decay (NMD), premature termination codon (PTC), frameshift or truncation

Amino Acid Substitution(s)

Displays whether an amino acid exchange occurs.

Within Protein Domains

Indicates whether the variant is located within a protein domain.

Altered Splicing

Indicates whether the variant leads to the alteration of a splice site.

Kozak Sequence Altered

Indicates whether the variant leads to the alteration of a Kozak sequence.

PolyA Signal Changed

Indicates whether the variant leads to the alteration of a poly-A signal.

miRNA Binding Site

Indicates whether the variant leads to the alteration of a miRNA binding site.

Open Chromatin

Indicates whether the variant is located within an open chromatin section.

Enhancer

Indicates whether an enhancer is annotated for the variant.

Promoter

Indicates whether the variant is located within a promoter. Active signifies that in addition, H3K4me3 and DHS annotations are also available for the site (please refer to our detailed page documentation for more information)

H3K4me3

Indicates whether the variant is located within a H3K4me3 positive region indicative for active transcription. Robust indicates that these annotations were available for at least three different cell lines.

Histone Modifications

Histone modification annotations from Ensembl at the site of interest.

Within TFBS

Indicates whether the variant is located within a transcription factor binding site (TFBS) from Ensembl [2].

Polymerase

Polymerase II and III binding sites annotated for the region.

Genomic Interaction(s)

Indicates whether the variant is located within a genomic interaction site according to Rao et al [1]

PhyloP /PhastCons (max)

Indicates the highest PhyloP [3] and PhastCons [4] scores, respectively.

CADD (Scaled)

Indicates the scaled CADD score for the alteration.

Variant Frequency

Variant frequency in dbSNP and 1000G. Unknown/rare alleles are marked with a bright red colour.

Output: detailed

Contact

References