Preliminary notes {#Preliminary}

The Shiny Variant Explorer (tSVE) was primarily developped to demonstrate features implemented in the r Biocpkg("TVTB"), not as a production environment. As a result, a few important considerations should be made to clarify what should and should not be expected from the web-application:

[^1]: The ... argument is called "ellipsis".

Pre-requisites {#Prerequisites}

The Shiny Variant Explorer suggests a few additional package dependencies compared to the package, to support certain forms of data input and display.

Input

[^2]: In the future, the web-application may also support TxDb and OrganismDb annotation packages.

[^3]: In the future, the web-application may also use annotation packages to facet statistics and figures by genomic range(s).

Display

Launching the Shiny Variant Explorer {#Launch}

The TVTB::tSVE() method launches the web-application.

Overall layout of the web-application {#OverallLayout}

Overall, the web-application is implemented as a web-page with a top level navigation bar organised from left to right to reflect progression through a typical analysis, with the exception of the last two menu items Settings and Session, which may be useful to check and update at any point.

Here is a brief overview of the menu items:

Input panel {#Input}

The Input panel controls the major input parameters of the analysis, including phenotypes (and therefore samples), genomic ranges, and fields to import from VCF file(s). Those inputs are useful to import only data of interest, as well as to limit memory usage and duration of calculations.

Phenotypes {#InputPhenotypes}

Phenotypes are critical to define groups of samples that may be compared in summary statistics, tables, and plots. Moreover, phenotypes also implicitely define the set of samples required in the analysis (unique sample identifiers usually set as rownames of the phenotypes).

The web-application accepts phenotypes stored in a text file, with the following requirements:

When provided, phenotypes will be used to import from VCF file(s) only genotypes for the corresponding samples identifiers. Moreover, an error message will be displayed if any of the sample identifiers present in the phenotypes is absent from the VCF file(s).

Note that the web-application does not absolutely require phenotype information. In the absence of phenotype information, all samples are imported from VCF file(s).

Action:

  • Click on the Browse action button
  • Navigate to the extdata folder of the TVTB installation directory
  • Select the file integrated_samples.txt

Alternatively: click the Sample file button

Notes

system.file("extdata", package = "TVTB")

Genomic ranges {#InputGRanges}

Genomic ranges are critical to import only variants in targeted genomic regions or features (e.g. genes, transcripts, exons), as well as to limit memory usage and duration of calculations.

The Shiny Variant Explorer currently supports three types of input to define genomic ranges:

Currently, the web-application uses genomic ranges solely to query the corresponding variants from VCF file(s). In the future, those genomic ranges may also be used to produce faceted summary statistics and plots.

Notes:

BED file {#InputBED}

If a BED file is supplied, the web-application parses it using the r Biocpkg("rtracklayer") import.bed method. Therefore the file must respect the BED file format guidelines.

Action:

  • Click on the Browse action button
  • Navigate to the extdata folder of the TVTB installation directory
  • Select the file SLC24A5.bed

Alternatively: click the Sample file button

Notes:

UCSC format {#InputUCSC}

Sequence names (i.e. chromosomes), start, and end positions of one or more genomic ranges may be defined in the text field, with individual regions separated by ";".

Action:

  • Paste 15:48,413,169-48,434,869 in the text field

Alternatively: click the Sample input button

Notes:

Ensembl-based annotation packages {#InputEnsDb}

Currently, genomic ranges encoding only gene-coding regions may be retrieved from an Ensembl-based database. This feature was adapted from the web-application implemented in the r Biocpkg("ensembldb") package.

\bioccomment{ In the future, the interface to query transcripts and exons annotations may be added to the web-application. }

Action:

  • Paste SLC24A5 in the text field

Alternatively: click the Sample input button

\fixme{ Genomic feature located on contigs may cause problems when working with one VCF per chromosome. In the future, an option may be added to ignore contigs. }

Variants {#InputVariants}

At the core of the r Biocpkg("TVTB") package, variants must be imported from one or more VCF file(s) annotated by the Ensembl Variant Effect Predictor (VEP) script [@RN1].

Considering the large size of most VCF file(s), it is common practice to split genetic variants into multiple files, each file used to store variants located on a single chromosome (more generally; a single sequence). The Shiny Variant Explorer supports two situations:

In addition, VCF files can store a plethora of information in their various fields. It is often useful to select only a subset of fields relevant for a particular analysis, to limit memory usage. The web-application uses the r Biocpkg("VariantAnnotation") scanVcfHeader to parse the header of the VCF file (Single-VCF mode) or the first VCF file (Multi-VCF mode), to display the list of available fields that users may choose to import. A few considerations must be made:

Single-VCF mode {#SingleVCF}

This mode display an action button that must be used to select the VCF file from which to import variants.

Action:

  • Click on the Browse action button
  • Navigate to the extdata folder of the TVTB installation directory
  • Select the file chr15.phase3_integrated.vcf.gz

Alternatively: click the Sample file button

Multi-VCF mode {#MultiVCF}

This mode requires two pieces of information:

Note that a summary of VCF file(s) detected using the given the folder and pattern is displayed on the right, to help users determine whether the parameters are correct. In addition, the content of the given folder is displayed at the bottom of the page, beside the same content filtered for the VCF file naming pattern.

Action:

None. The text fields should already be filled with default values, pointing to the single example VCF file (chr15.phase3_integrated.vcf.gz).

VCF scan parameters {#scanVcfParam}

This panel allows users to select the INFO and FORMAT fields to import (in the info and geno slots of the VCF object, respectively).

It is important to note that the FORMAT/GT and INFO/ fields---where <vep> stands for the INFO key where Ensembl VEP predictions are stored---are implicitely imported from the VCF. Similarly, the mandatory FIXED fields CHROM, POS, ID, REF, ALT, QUAL, and FILTER are automatically imported to populate the rowRanges slot of the VCF object.

Action:

  • Click the Deselect all action button under the INFO fields selection input to import only the INFO/CSQ and FORMAT/GT fields.
  • Click the Import variants action button

A summary of variants, phenotypes, and samples imported will appear beside the action button.

Annotations {#EnsDbPkg}

This panel allows users to select a pre-installed annotation package. Currently, only EnsDb annotation packages are supported, and only gene-coding regions may be queried.

Action:

  • If none of the EnsDb packages are installed, it will simply not be possible to use the ensembl interface of the Genomic ranges input tab.
  • If the EnsDb.Hsapiens.v75 package is the only EnsDb packages installed, no action is required; the package should already be pre-selected.
  • If the EnsDb.Hsapiens.v75 package is not the only EnsDb packages installed, users should select it in the list of choices.

Frequencies panel {#Frequencies}

This panel demonstrates the use of three methods implemented in the r Biocpkg("TVTB") package, namely addFrequencies, addOverallFrequencies, and addPhenoLevelFrequencies.

Overall frequencies {#OverallFrequencies}

This panel allows users to Add and Remove INFO fields that contain genotype counts (i.e. homozygote reference, heterozygote, homozygote alternate) and allele frequencies (i.e. alternate allele frequency, minor allele frequency) calculated across all the samples and variants imported. The web-application uses the homozygote reference, heterozygote, and homozygote alternate genotypes defined in the Advanced settings panel.

Importantly, the name of the INFO keys that are used to store the calculated values can be defined in the Advanced settings panel.

Action:

  • Click the Add action button
  • See the Latest changes message update at the top of the screen.
  • Optionally, the Views panel can be used to examine the new fields

Phenotype-level frequencies {#PhenoLevelFrequencies}

This panel allows users to Refresh the list of INFO fields that contain genotype counts and allele frequencies calculated within groups of samples associated with various levels of a given phenotype.

Action:

  • Select super_pop in the list of phenotypes
  • Click the Select all action button
  • Click the Refresh action button
  • See the Latest changes message update at the top of the screen.
  • Optionally, the Views panel can be used to examine the new fields

Filters panel {#VcfFilterRules}

One of the flagship features of the r Biocpkg("TVTB") package are the VCF filter rules, extending the r Biocpkg("S4Vectors") FilterRules class to new classes of filter rules that can be evaluated within environments defined by the various slots of VCF objects.

Generally speaking, FilterRules greatly facilitate the design and combination of powerful filter rules for table-like objects, such as the fixed and info slots of r Biocpkg("VariantAnnotation") VCF objects, as well as Ensembl VEP predictions stored in the meta-columns of GRanges returned by the r Biocpkg("ensemblVEP") parseCSQToGRanges method.

A separate vignette describes in greater detail the use of classes that contain VCF filter rules. A simple example is shown below.

Action:

  • Select VEP as the Type of filter
  • Paste grepl("missense",Consequence) in the text field
  • Leave the Active? checkbox ticked
  • Click the Add filter action button
  • See the list of rules update at the bottom of the screen
  • Click the Apply filters action button
  • See the summary of filtered variants update beside the action button
  • Optionally, the Views panel can be used to examine the new fields

Alternatively: click the Sample input button

Views panel {#Views}

This panel offers the chance to examine the main objects of the session, namely:

Action:

  • In the various panels, select fields to examine each object
    • In particular, note the INFO fields that contain genotype counts and allele frequencies calculated earlier
  • Go to the Heatmap tab of the Genotypes panel
  • Click the Go! action button to calculate and display the heatmap

Plots panel {#Plots}

This panel demonstrates the use of two methods implemented in the r Biocpkg("TVTB") package, namely tabulateVepByPhenotype and densityVepByPhenotype.

Settings panel

This panel stores more advanced settings that users may not need to edit as frequently, if at all. Those settings are divided in two sub-panels:

Advanced settings {#AdvancedSettings}

Genotypes {#AdvancedGenotypes}

It is critical to accurately identify and define how the different genotypes---homozygote reference, heterozygote, and homozygote alternate---are encoded in the VCF file, to produce accurate genotypes counts and frequencies, for instance. This generally requires examining the content of the FORMAT/GT field outside of the web-application. For instance, the functions unique and table may be used to identify (and count) all the distinct genotype codes in the geno slot ("GT" key) of a VCF object.

The default selected values are immediately compatible with the demonstration data set. Users who wish to select genotypes codes not yet available among the current choices may either contact the package maintainer to add them in a future release, or edit the Global configuration file of the web-application locally.

INFO key suffixes {#AdvancedSuffixes}

Currently, the three calculated genotypes counts and two allele frequencies require five INFO fields to store their respective values.

Considering that r Biocpkg("TVTB") offers the possibility to calculate counts and frequencies for the overall data set, and for each level of each phenotype, it is important to define a clear and consistent naming mechanism that does not conflict with INFO keys imported from the VCF file(s). In the r Biocpkg("TVTB") package, a suffix is required for each type of genotype and frequency calculated, to generate INFO as follows:

Again, the default values are immediately compatible with the demonstration data set. For other data sets, it may be necessary to change those values, either by preference, or to avoid conflict with INFO keys imported from the VCF file(s).

Miscellaneous settings {#AdvancedMiscellaneous}

Other rarely used settings in this panel include:

Parallel settings {#ParallelSettings}

Several functionalities of the r Biocpkg("TVTB") package are applied to independent subsets of data (e.g. counting genotypes in various levels of a given phenotype). Such processes can benefit from multi-threaded calculations. Multi-threading settings in the Shiny web-application are somewhat experimental, as they have been validated only on a small set of operating systems, while some issues have been reported for others.

| Report | Operating System | Cluster Class | Cluster type | # Cores | | :----: | :---:| :-----------: | :----------: | :-----: | | OK | Ubuntu 14.04 | Multicore | FORK | 2 | | OK | Scientific Linux 6.7 | Multicore | FORK | 2 | | Hang~1~ | OS X El Capitan | Snow | SOCK | 2 |

  1. Application hangs while CPUs work infinitely at full capacity.

\bioccomment{ Users are welcome to send feedback to report additional successful configuration, as well as newly identified issues. }

Session information {#SessionInfo}

The last panel of the Shiny Variant Explorer offers detailed views of objects and settings in the current session, including:

Global configuration {#GlobalConfig}

Most default values are stored in the global.R file of the web-application. All the files of the web-application are stored in the extdata/shinyApp folder of the r Biocpkg("TVTB") installation directory (see an earlier section to identify this directory).

Users who wish to change the default values of certain input widgets (e.g. genotype codes) may edit the global.R file accordingly. However, the file will be reset at each package update.

\bioccomment{ In the future, a mechanism may be implemented to override global settings locally, without risk of seeeing this custom configuration overwritten at the next package update (e.g. a file in the user home folder that would be parsed to overwrite certain settings). }

Vignette session {#VignetteSessionInfo}

Here is the output of sessionInfo() on the system on which this document was compiled:

sessionInfo()

References {#References}



kevinrue/tSVE documentation built on Aug. 11, 2017, 4:14 p.m.