BaseSpace VariantStudio User Guide - Support

BaseSpace VariantStudio User Guide - Support

BaseSpace VariantStudio

™ v2.2

Software User Guide

FOR RESEARCH USE ONLY

ILLUMINA PROPRIETARY

Part # 15047059 Rev. A

June 2014

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Part # 15047059 Rev. A

Revision History

Part #

15047059

Revision

A

Date

June 2014

Description of Change

Initial release.

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Part # 15047059 Rev. A

Table of Contents

Revision History

Table of Contents

Chapter 1 Getting Started

Introduction

VCF Input Requirements

BaseSpace VariantStudio Software Interface

Create or Open a Project

Import Variant Call Files

Data in BaseSpace VariantStudio Tables

VCF Fields Reported in the Variants Table

Somatic VCF Fields Reported in the Variants Table

Chapter 2 Applying Annotations and Classifications

Annotate Variants

Create Custom Annotations

Apply Variant Classifications

Edit Variant Classifications

Manage Classifications

Import Classifications

Chapter 3 Applying Filters

Apply Filters

Family-Based Filtering Workflows

Create Favorite Filters

Chapter 4 Generating Reports

Introduction

Sample Report Overview

Create a Sample Report Template

Create a Sample Report

Export Text Files and Charts

Appendix A Annotation Sources

Annotation Sources

Index

Technical Assistance

v

vii

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12

21

22

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9

2

4

25

26

29

31

33

35

37

39

40

49

55

57

58

59

60

62

63

65

66

69

71

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  Chapter 1  Getting Started

Getting Started

Introduction

VCF Input Requirements

BaseSpace VariantStudio Software Interface

Create or Open a Project

Import Variant Call Files

Data in BaseSpace VariantStudio Tables

VCF Fields Reported in the Variants Table

Somatic VCF Fields Reported in the Variants Table

9

10

12

21

22

2

4

6

BaseSpace VariantStudio v2.2 Software User Guide

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Introduction

The BaseSpace VariantStudio™ software is an executable that gets installed on your desktop computer when you launch the BaseSpace VariantStudio App. BaseSpace

VariantStudio imports SNPs and indels from your projects in BaseSpace or BaseSpace

Onsite. After import, BaseSpace VariantStudio provides commands to annotate variants, filter results using filtering options, and export data to a report.

Figure 1 BaseSpace VariantStudio Workflow

Launch the BaseSpace VariantStudio App and associate to a project.

Import SNPs and indels from BaseSpace or BaseSpace Onsite. Import one sample or multiple samples to a single project.

Annotate variants for the current sample. An internet connection is required to annotate variants.

Filter data based on any combination of filtering options. Save filter combinations for use in other projects.

Classify variants according to their biological impact.

Generate a sample report using a customized report template.

Export data and filtering history to text files. Export data to a histogram or pie chart.

NOTE

When you have already installed the BaseSpace VariantStudio App, it is best to launch

BaseSpace VariantStudio from the desktop instead of going through BaseSpace or BaseSpace

Onsite.

System Requirements

Installing the BaseSpace VariantStudio software requires the following system specifications:

}

64-bit Windows OS (Windows 7, or later)

}

2 GB RAM minimum; 4 GB RAM recommended

}

25 MB hard drive space for installation

} Internet connection required

NOTE

An internet connection is required for annotating variants. After variants have been annotated and saved in a project, an internet connection is no longer required.

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Installation

To install BaseSpace VariantStudio, launch the BaseSpace VariantStudio App from your account in BaseSpace or BaseSpace Onsite. For instructions, see the BaseSpace User Guide or

BaseSpace Onsite User Guide.

NOTE

Installation of VariantStudio v2.2, or later, does not overwrite the version previously installed on your computer, allowing side-by-side installation of different software versions.

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VCF Input Requirements

BaseSpace VariantStudio imports SNPs and indels reported in VCF v4.0, or later, file formats. If analysis software other than Illumina analysis software is used to generate data, the VCF file might not contain the required columns.

VCF Column

CHROM

POS

ID

REF

ALT

QUAL

INFO

Required Value

The chromosome number. Values are #, c#, or chr#, where # is the chromosome number, as in 1–22, or name, as in X or Y, or M for mitochondrial.

The position of the variant. Values are numeric with the first base having position 1 (1-based).

The ID is the rs number for the SNP as recorded in dbSNP.txt. A value must be present. If a dbSNP entry does not exist, a missing value marker '.' is an acceptable value.

Although the ID column and valid values are required, the values are not imported. The software applies dbSNP annotations with the Annotate command.

The reference allele.

The alternate allele.

The quality score assigned by the variant caller. A value of '.' is acceptable, and is reported as a 0.

Recognized fields are VF (alt variant freq), DP (read depth), AD (allelic depth), SOMATIC, and '.' (none).

• VF—Represented in the Alt Variant Freq column in the Variants table.

• DP—Represented in the Read Depth column in the Variants table.

• DPI—Represented in the Read Depth column in the Variants table for insertion and deletion events called by the Illumina Isaac Alignment and

Variant Calling workflow.

• AD—Represented in the Alt Read Depth and Allelic Depth columns in the Variants table.

• SOMATIC—Represented in the Genotype column in the Variants table.

This value applies only to somatic variants.

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VCF Column

FORMAT

Required Value

A list of fields that define values in the Sample column. Possible values are

VF (alt variant frequency), DP (read depth), AD (allelic depth), GT

(genotype), and '.' (none).

• VF—Represented in the Alt Variant Freq column in the Variants table.

• DP—Represented in the Read Depth column in the Variants table.

• DPI—Represented in the Read Depth column in the Variants table for insertion and deletion events called by the Illumina Isaac Alignment and

Variant Calling workflow.

• AD—Represented in the Alt Read Depth and Allelic Depth columns in the Variants table.

Genotype Values: (Not present in somatic VCF files.)

• Acceptable GT values are 0/0, 0/1, and 1/1. Non-numeric GT values, or

'./.' as in a no-call, are not imported.

• Hemizygous alt GT values, '1', are accepted. Hemizygous reference calls,

'0', are not imported.

• If FORMAT and Sample are not empty, then a GT value is required.

• IF FORMAT and Sample are empty, the software assumes that GT is heterozygous, 0/1.

Genome VCF Files

Importing genome VCF (gVCF) files is supported as of BaseSpace VariantStudio v2.1 for targeted enrichment data.

Using gVCF is not recommended for whole genomes without pre-processing with gVCF tools. Alternatively, you can load only exonic regions, or only regions from a gene list or

BED file without the need for pre-processing. For more information, see

VCF Import Options

on page 10

.

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BaseSpace VariantStudio Software Interface

When the BaseSpace VariantStudio software launches, the interface opens with BaseSpace

File Browser. This file browser allows you to import a BaseSpace VCF file to VariantStudio.

Figure 2 BaseSpace File Browser

Interface Commands

The BaseSpace VariantStudio interface is an interactive view of genes and variants in a selected sample. Use the interface commands to import VCF files, sort data, apply filters, and export data to a report.

Figure 3 BaseSpace VariantStudio Interface

6

A

Menu and commands—Contains commands for managing the project, annotating variants, and reporting results. Commands are organized in four tabs: Home,

Annotation and Classification, Reports, and Help.

B

Filters pane—Provides options for filtering data using any combination of filters.

C

Filter history—Opens the history panel that shows all filters applied to the project.

D

Table tabs—Navigation between the Variants table, Genes table, and No-Call Regions table.

E

Gene view—Shows a graphical representation of the selected gene.

F

Table views—View of data shown in the Variants table, Genes table, and No-Call

Regions table. Use the table tabs to toggle between table views.

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Filters Pane

The Filters pane provides various filtering options to narrow results to your area of interest.

Combine any number of filtering options from the filter categories and click Apply Filters.

Filters are applied to the current sample only, not to all samples that are imported into the project. To clear filters, click Clear Filters. For more information, see

Apply Filters on page

40 .

Figure 4 Filters Pane

Filter History

The Filter History pane shows filters that have been applied to the samples in this project.

Filters can be a single filter, a combination of filters, or a saved filter from the favorite filter list.

Figure 5 Filter History Pane

Column Heading

Num Genes

Num Variants

Filter Name

Filter

Description

The number of genes showing with the filters applied.

The number of variants showing with the filters applied.

The name of the filter applied. The filter name appears only if the filter was saved as a favorite. Otherwise, the filter name is

Untitled.

The description of the filter applied, which can describe one filter or a combination of filters.

The Filter History pane includes three buttons: Clear History, View, and Apply:

} Clear History—Clears entries in the filter history pane.

}

View—Shows a block diagram illustration of the filter.

}

Apply—Applies the filter to the variants table.

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You can export information from the Filter History pane in a comma-separated values

(CSV) file format. For more information, see

Generating Reports on page 57 .

Gene View

The Gene View shows a graphical representation of the gene with the following indicators:

} Exons are indicated in dark blue.

} Variants are indicated with a red line.

}

The selected variant is indicated with an orange line.

}

The selected transcript is indicated in purple.

} No-call regions are indicated in gray.

Figure 6 Gene View

The Gene View is interactive. Using your mouse, hover over the view to see the coordinate.

Click and drag your mouse to slide the view from end to end. Use the scrolling feature on your mouse to zoom in and zoom out.

Menus and Commands

BaseSpace VariantStudio commands are arranged in the following four tabs:

} Home tab—Contains commands for saving projects, importing data, managing favorite filters, and changing layout options. For more information, see the following sections:

Import Variant Call Files on page 10

Modify Table Views on page 18

Create Favorite Filters on page 55

}

Annotation and Classification—Contains commands to annotate variants and apply classifications. For more information, see

Annotate Variants on page 26 .

}

Reports tab—Contains commands for exporting results to reports. For more information, see

Introduction on page 58

.

} Help tab—Contains information about the software version and a link to online help.

An internet connection is required to access the help files.

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Create or Open a Project

The Project menu includes commands to create, open, save, and name projects.

Figure 7 Project Menu Commands

Command

New

Open

Close

Save

Save As

Description

Creates a project. Starting a new project closes the current project.

If you have not yet saved changes to the current project, a reminder to save your changes appears.

Opens a project. Opening another project closes the current project.

If you have not yet saved changes to the current project, a reminder to save your changes appears.

Closes the current project. This command does not close the software application.

If you have not yet saved changes to the current project, a reminder to save your changes appears.

Saves changes made to an open project. If your project is new, you are prompted to name the project.

Save As...: Provides the option to save the current project with a different name.

Save As Reduced Project: Provides the option to save the current project with a different name, at a reduced size. You cannot recover variants that were filtered out when saving at a reduced size.

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Import Variant Call Files

From the Home tab, use commands on the Samples menu to import variant call files in

VCF file format and manage samples in the project.

Figure 8 Samples Menu Commands

Command

Import VCF

Add Variants to

Sample

Import Folder

Current Sample

Remove Sample

Description

Opens a window to browse to a file location and import one selected VCF file.

Opens a window to browse to a file location and import SNPs and indels from another VCF file. This command imports data from the selected VCF file and adds it to the current sample.

Important: There is no change to the sample name to denote that variant calls have been merged.

Opens a window to browse to a folder location and import all

VCF files in the selected folder.

Shows the active sample name. The Current Sample dropdown list shows all samples in the project. To change to another sample in the project, select a sample name from the drop-down list.

Removes the current sample from the project. A confirmation dialog box opens before the sample is removed from the project.

VCF Import Options

With any command to import variant calls, the VCF Import Options dialog box opens.

From this dialog box, specify which variants to import using one of four options.

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Figure 9 VCF Import Options

Command

All variants

Variants in exons

Variants specified in a gene list

Variants in regions specified in a BED file

Load hom-ref positions

Description

Select the radio button to import all variants in the selected

VCF files.

Select the radio button to import only variants found in exonic regions. With this option, set the number of bases, or padding, to include on both sides of the exon.

Select the radio button and click Browse to navigate to the location of a gene list file. A gene list file must be a text file with a *.txt file extension that lists one gene per line.

Select the radio button and click Browse to navigate to the location of the BED file.

This setting applies to gVCF files.

Select the checkbox to import all homozygous reference positions, 0/0.

Clear the checkbox to omit homozygous reference positions from the import.

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Data in BaseSpace VariantStudio Tables

Imported and annotated information for the visible sample is arranged in three tables on the BaseSpace VariantStudio interface: Variants table, Genes table, and No-Call Regions table. Use the tabs below the table area to navigate between tables.

Figure 10 Navigation Tabs for Variants Table, Genes Table, and No-Call Regions Table

Variants Table

The Variants table lists the genes that overlap variants identified in the selected sample.

Each row of the table contains the gene and reported variant. Genes that include multiple variants are listed multiple times in the table, one time for each variant.

The following information is provided in the Variants table. If a column described in the following is not visible in your instance of BaseSpace VariantStudio, click Column Order from the Table Options menu to view hidden columns.

Column Heading

Gene

Variant

Chr

Coordinate

Classification

Type

Description

The name of the gene.

Lists the reference allele and the diploid genotype call for the sample as Reference > AlleleA/AlleleB.

AlleleA and AlleleB are explicitly defined from the REF, ALT, and

GT fields of the VCF file. For example, at a heterozygous position noted as GT=0/1 is represented as REF > REF/ALT, and a homozygous non-reference position noted as GT=1/1 is represented as REF > ALT/ALT.

The chromosome number in which the gene occurs.

The genomic location of the variant (1-based).

The classification assigned to the variant. This field is populated for variants that match criteria specified in the classification database.

The type of variant, which is either a single nucleotide variant

(SNV), insertion, deletion, or ref (for reference call).

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Column Heading

Genotype

Exonic

Filters

Quality

GQX

Inherited From

Alt Variant Freq

Read Depth

Alt Read Depth

Allelic Depth

Custom Annotation

Custom Gene

Annotation

Custom Gene

Annotation 2

Num Transcripts

Transcript

Description

The genotype, which is either heterozygous (het), homozygous

(hom) or somatic (som).

A variant found within a coding region, ±20 bp on both sides of the coding region.

The status of the variant call quality as annotated in the VCF file.

PASS indicates that all filters were passed; otherwise the variant call filter is listed.

The filter listed and threshold for passing filter depends on the method used to generate the VCF file.

The numeric value of variant call quality as written to the QUAL column of the VCF file. Determination of variant quality depends on the variant caller.

The conservative measure of genotype quality derived from the minimum of the GQ and QUAL values listed in the VCF file.

This field is not populated for somatic VCF files. For more information, see

Somatic VCF Fields Reported in the Variants Table on page 22 .

The inherited source of the variant. Possible values are father, mother, both, indeterminate, or ambiguous.

If the variant is heterozygous in the father, mother, and child, a variant is listed as ambiguous.

If the variant is homozygous in the child and heterozygous in both parents, a variant is listed as both.

If the inheritance of the variant cannot be determined from the other VCFs, the variant is listed as indeterminate.

Entries in this column are meaningful only when the family-based filter is applied.

The frequency of the Alt Allele.

The total number of reads passing quality filters at this position.

The number of reads called at this position.

The number of reads called for the Ref Allele and the Alt Allele.

Annotations according to values provided in the Annotation column of an optional custom annotation file.

Annotations according to values provided in the Annotation column of an optional custom gene annotation file.

Annotations according to values provided in the Gene Annotation

2 column of an optional custom annotation file.

The number of transcripts reported in the annotation, which includes overlapping transcripts and upstream and downstream transcripts within 5 kb of the variant.

The name of the transcript, usually a database identifier from

RefSeq or Ensembl.

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Column Heading

Consequence cDNA Position

CDS Position

Protein Position

Amino Acids

Codons

HGNC

Transcript HGNC

Canonical

SIFT

PolyPhen

ENSP

HGVSc

HGVSp dbSNP ID

Ancestral Allele

Allele Freq

Allele Freq Global

Minor

Global Minor Allele

Allele Freq Amr

Allele Freq Asn

Allele Freq Af

Allele Freq Eur

Allele Freq Evs

Description

Consequence of the variant, described in Sequence Ontology standardized vocabulary.

Position of the variant in cDNA.

Position of the variant in the coding region.

Position of the amino acid in the protein

Amino acid or amino acid change. If the variant is synonymous, then there is no change and one amino acid is listed.

Specific codon noted with and without the variant, highlighted in uppercase.

The gene name, expressed as official HGNC nomenclature.

The transcript name, expressed as official HGNC nomenclature.

Indicates whether the transcript is the canonical transcript.

SIFT score.

PolyPhen score.

Protein ID (Ensembl ID).

Human Genome Variation Society (HGVS) notation in the cDNA.

Human Genome Variation Society (HGVS) notation in the protein.

The rsID entry in dbSNP.

The inferred allele ancestral to the chimpanzee/human lineage. For more information, see www.1000genomes.org/faq/where-doesancestral-allele-information-your-variants-come.

The allele frequency from all populations of 1000 genomes data;

April 2012 phase 1 call set (v3 update).

Global minor allele frequency (GMAF); technically, the frequency of the second most frequent allele. For more information, see www.ncbi.nlm.nih.gov/projects/SNP/docs/rs_ attributes.html#gmaf.

The specific allele with the reported GMAF.

The allele frequency from 1000 Genomes (Ad Mixed American population).

The allele frequency from 1000 Genomes (East Asian population).

The allele frequency from 1000 Genomes (African population).

The allele frequency from 1000 Genomes (European population).

The allele frequency from the NHLBI exome sequencing project.

Exome Variant Server, NHLBI GO Exome Sequencing Project

(ESP), Seattle, WA (evs.gs.washington.edu/EVS) [November 2012 accessed]

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Column Heading

EVS Coverage

EVS Samples

Conserved Sequence

COSMIC ID

COSMIC Wildtype

COSMIC Allele

COSMIC Gene

COSMIC Primary Site

COSMIC Histology

ClinVar Accession

ClinVar Ref

ClinVar Alleles

ClinVar Allele Type

ClinVar Significance

Regulatory Feature

Alternate Alleles

Google Scholar

Description

The average depth of coverage for SNVs that were called at this position from the Exome Variant Server (EVS).

The number of samples that were called at this position from the

Exome Variant Server (EVS).

Denotes if the variant is an identical or similar sequence that occurs between species and maintained between species throughout evolution.

The numeric identifier for the variant in Catalogue of Somatic

Mutations in Cancer (COSMIC) database, if the genomic position of the variant overlaps a variant listed in COSMIC.

The COSMIC ID links to the COSMIC page associated with the identifier.

The allele in unaffected individuals as reported in the COSMIC database.

The allele as reported in the COSMIC database.

The gene name as reported in the COSMIC database.

The primary tissue type associated with the allele as reported in the COSMIC database.

The tissue type associated with the allele as reported in the

COSMIC database.

The alpha-numeric ID assigned to the allele in the ClinVar database and link to the associated page of the ClinVar database.

The Reference Allele as reported in the ClinVar database.

The name of the allele as reported in the ClinVar database.

The type of allele, either single nucleotide variant (SNV), insertion, deletion, or duplication as reported in the ClinVar database.

The clinical significance or classification assigned to the allele as reported in the ClinVar database.

A link to regulatory information in Ensembl for that genomic region.

The number of nucleotide bases called for Allele A and Allele B that differ from the RefAllele.

Link to the Google Scholar search page for the selected variant, cDNA, and amino acid. The page opens with an auto-populated search field. Click search to continue.

The search is transcript-dependent. If a rs number is present, the number is included in the search.

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Column Heading

PubMed

UCSC Browser

ClinVar RS

ClinVar Disease

Name

ClinVar MedGen

ClinVar OMIM

ClinVar Orphanet

ClinVar Gene

Reviews

ClinVar SnoMedCt ID

Exon

Intron

Distance

Description

Link to PubMed search page for the selected variant, cDNA, and amino acid. The page opens with an auto-populated search field.

Click search to continue.

The search is transcript-dependent. If a rs number is present, the number is included in the search.

Link to UCSC Browser search page for the selected chromosome and position.

The numeric rsID assigned to the allele in the ClinVar database.

The disease associated with the allele as reported in the ClinVar database.

The alpha-numeric identifier of the disease as reported by MedGen and link to the associated page of the MedGen database.

The numeric identifier for the disease as reported by Online

Mendelian Inheritance in Man (OMIM) and link to the associated page of the OMIM database.

The numeric identifier of the disease as reported by Orphanet and link to the associated page in the Orphanet database.

The alpha-numeric identifier of the disease as reported by Gene

Reviews and link to the associated page in the Gene Review database.

The numeric identifier of the disease and associated clinical terms as reported by SnoMedCt.

The exon number in which the variant is present.

The intron number in which the variant is present.

Distance between the variant and the nearest end of the gene.

• For upstream variants, this value is the distance to the beginning of the first exon.

• For downstream variants, this value is the distance to the end of the last exon.

Genes Table

The Genes table lists the genes that contain variants identified in the selected sample. Each row of the table contains the gene and number of variants reported, along with the following information reported for each gene.

Column Heading

Name

Gene ID

Chr

Description

The name of the gene.

The Entrez Gene ID for the gene and link to the associated entry in the NCBI database.

The chromosome number in which the gene occurs.

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Column Heading

Start

Stop

Length

Num Alleles

Num Variants

Paternal Variants

Maternal Variants

Ambiguous Variants

Custom Gene

Annotation

Custom Gene

Annotation 2

PubMed

GeneReviews

Disease

Description

The start coordinate of the gene (1-based).

The end coordinate of the gene.

The length of the gene.

The number of alleles reported.

The number of variants reported.

The number of variants inherited from the father.

The number of variants inherited from the mother.

The number of ambiguous variants.

Gene annotations according to values provided in the Annotation column of an optional custom gene annotation file.

Annotations according to values provided in the Gene Annotation

2 column of an optional custom annotation file.

Link to PubMed search page for the selected gene. The page opens with an auto-populated search field. Click search to continue.

Link to GeneReviews website. Clicking the entry provides a dropdown list with links to GeneReviews.

Diseases associated with the gene. Clicking the disease name provides a drop-down list with links to MedGen and OMIM.

Description The protein name associated with gene function.

No-Call Regions Table

The No-Call Regions table shows regions where calls could not be confidently made due to a low read depth or failing a quality filter. The No-Call Regions table is populated directly from the non-variant regions reported in the genome VCF (gVCF) file. Non-variant regions are reported as 0/0 in the gVCF file. For more information, see sites.google.com/site/gvcftools/home/about-gvcf/gvcf-conventions

Each row of the table contains the gene and information reported for the gene. The following information is provided in the No-Call Regions table.

Column Heading

Gene

Chr

Start

Description

The name of the gene located within the no-call region.

The chromosome number in which the no-call region occurs.

The start chromosomal coordinate of the no-call region.

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Column Heading

Stop

Length

Depth

Quality

Filter

Description

The end chromosomal coordinate of the no-call region.

The length of the no-call region.

The read depth of the no-call region.

The numeric value of variant call quality as written to the QUAL column of the VCF file. Determination of variant quality depends on the variant caller.

The filter associated with the variant call quality as annotated in the

VCF file.

Modify Table Views

To modify how data appear in the tables, click the column headings. Options include sorting in descending or ascending order, showing only selected data based on listed values, or adjusting column order.

NOTE

Modifying how data appear in the Variants table only affects how information is arranged in the table. Modifying views does not change the underlying data.

Sort Data in Ascending or Descending Order

To change the order in which data appear in the Variants table, click a column heading.

Data are sorted in either descending or ascending order of values listed in that column.

Click again to reverse the order.

} When the table is sorted in ascending order, the up arrow icon appears in the column heading.

}

When the table is sorted in descending order, the down arrow icon appears in the column heading.

Show Only Selected Data

To show only selected data based on information in the Variants table, use the show/hide icon in the column heading.

1 Click the show/hide icon in the column heading. A drop-down list opens that contains all values present in that column.

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2 Select a value from the drop-down list. The Variants table shows only data that contains your selection.

To restore the default view of the Variants table, use one of the following methods:

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}

Click the show/hide icon in the column heading used to modify the table and select

All from the drop-down list.

}

Click the icon at the bottom of the Variants table.

A history of previous selections appears at the top of the column heading drop-down list for quick access to frequently used selections. To remove a selection from history, click the delete icon.

Show or Hide Selected Columns

Use the checkboxes below the table tabs to show or hide specific columns in the Variants table. Select the checkbox to show data, and clear the checkbox to hide data. All options are set to show, by default.

Show/hide options include the following sections of the Variants table:

}

Show Population Frequencies—Shows and hides Allele Freq, Allele Freq Global Minor, global Minor Allele, Allele Freq Amr, Allele Freq Asn, Allele Freq Af, Allele Freq Eur, and Allele Freq Evs.

} Show Transcript Info—Shows and hides Num Transcripts, Transcript, Consequence, cDNA Position, CDS Position, Amino Acids, Codons, Exon, Intron, Transcript HGNC,

Distance, Canonical, Sift, PolyPhen, ENSP, HGVSc, and HGVSp.

}

Show Custom Annotations—Shows and hides Custom Annotation, Custom

Annotation 2, Custom Annotation 3, Custom Annotation 4, and Custom Gene

Annotation.

} Show ClinVar—Shows and hides ClinVar RS, ClinVar Ref, ClinVar Alleles, ClinVar

Significance, ClinVar Disease Name, ClinVar Accession, ClinVar MedGen, ClinVar

OMIM, ClinVar Orphanet, ClinVar Gene Reviews, and ClinVar SnoMedCt ID.

}

Show COSMIC—Shows and hides COSMIC ID, COSMIC Wildtype, COSMIC Allele,

COSMIC Gene, COSMIC Primary Site, and COSMIC Histology.

Set Table Options

Figure 11 Table Options Menu

The Table Options menu includes the following commands:

} Select All, which selects all rows in a table.

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} Copy, which copies selected data to the clipboard.

}

Smaller and Larger, which changes text size in a table.

}

Column Order, which includes commands to change table layout.

From the Table Options menu, click Column Order to open the Table Column Display window. From this window, drag and drop column headings to specify table layout:

1 To show or hide columns, drag and drop column headings from the Displayed

Columns list to the Hidden Columns list.

2 To prevent selected columns from scrolling horizontally, drag and drop column headings from the Scrolling list to the Fixed list.

3 Click OK when you are finished.

4 To save this layout for use in other BaseSpace VariantStudio projects, click Save As

Default from the Layout menu.

In the following example, the Gene column is set to Fixed, and variant length and optional custom annotation columns are hidden.

Figure 12 Table Column Display Window

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VCF Fields Reported in the Variants Table

Several columns of the Variants table are populated from columns or fields in the VCF file, as described in the following table.

Variants Table

Column Heading

Allelic Depth

VCF File Column or Field Description

Alt Read Depth

Alt Variant Freq

Chr

Coordinate

Exonic

Filters

Gene

Genotype

GQX

Quality

Read Depth

Type

Variant

Based on values listed for AD in INFO or FORMAT/[Sample

Name].

Based on the second value listed for AD in INFO or FORMAT/

[Sample Name].

Based on values listed for VF in INFO or FORMAT/[Sample

Name].

Based on values in the CHROM column.

Based on values in the POS column.

Based on values in the CHROM and POS columns, and calculated from a list of exonic regions.

Based on values in the FILTER column.

Based on values in the CHROM and POS columns, and calculated using a list of gene coordinates.

Based on values listed for GT in FORMAT/[Sample Name].

Based on values listed for GQX in FORMAT/[Sample Name].

Based on values in the QUAL column.

Based on values listed for DP in FORMAT/[Sample Name], DPI in

FORMAT/[Sample Name] for insertion and deletion events called by the Illumina Isaac Alignment and Variant Calling workflow.

Based on the number of bases in the REF and ALT columns.

Based on values in the REF and ALT columns.

• At a heterozygous position, the value is REF > REF/ALT.

• At a homozygous position, the value is REF > ALT/ALT.

NOTE

Some fields reported in the Variants table differ for somatic VCF files. For more information, see

Somatic VCF Fields Reported in the Variants Table on page 22

.

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Somatic VCF Fields Reported in the Variants Table

Information reported in BaseSpace VariantStudio for VCF files generated by the Illumina cancer analysis pipeline differs from what is reported for other VCF files.

For these files, there is no genotype (GT) or genotype score (GQX). Instead, allelic depths are listed.

Each VCF includes two samples, one of which is a reference and the other is the cancer sample. All reported values are specific to the cancer sample.

The following table lists the VCF fields that are unique to somatic VCF files.

Variants Table

Column Heading

Allelic Depth

Alt Read Depth

Alt Variant Freq

Genotype

VCF File Column or Field Description

Based on values in the FORMAT column. Allelic Depth is calculated differently for SNVs and indels:

For SNVs—Based on four values listed as AU:CU:GU:TU in the

FORMAT column. These values are listed as two numbers each, separated by a comma, and represent each possible allele in the cancer sample. The Allelic depth column is populated with the full set of numbers, 0,0:0,0:10,10:3,4.

For indels—Two values listed as TAR:TIR in the FORMAT column represent the Ref Allele and Alt Allele, respectively. Only the first number in each value is used. In the example 0,0:12,12, the Ref Allele is 0 and the Alt Allele is 12. Allelic Depth is listed as

0,12.

Based on values in the FORMAT column. Alt Read Depth is calculated differently for SNVs and indels:

For SNVs—Based on the first value from the appropriate Allelic

Depth (AU:CU:GU:TU). In the example 0,0:0,0:10,10:3,4, the values are 10,10 for GU and 3,4 for TU. If the Ref Allele is G and the Alt Allele is T, the Alt Read Depth is 3.

For indels—Based on the first value from the appropriate Allelic

Depth (TAR:TIR). In the example 0,0:12,12, the Ref Allele is 0 and the Alt Allele is 12. Alt Read Depth is listed as 12.

For somatic VCF files, allele frequency is calculated from values in the VCF file before data are reported in the Variants table.

For SNVs—Using only the first values for AU:CU:GU:TU, allele frequency is calculated as (alt allelic depth/(alt allelic depth + ref allelic depth))*100. In the example 0,0:0,0:10,10:3,4, Alt Variant

Freq is 23.08% by calculating (3/(3+10))*100.

For indels—Using only the first values for TAR and TIR, allele frequency is calculated as (TIR/(TIR+TAR))*100. In the example

0,0:12,12, Alt Variant Freq is 100% by calculating (12/(12+0))*100.

Based on values listed in the INFO column. If SOMATIC is listed in the INFO column, the genotype is listed as somatic (som) in the

Variants table.

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Variants Table

Column Heading

Quality

Read Depth

VCF File Column or Field Description

Quality is based on different values for SNVs and indels:

For SNVs—Quality is based on the QSS_NT field in the INFO column. This score represents the probability that the SNV exists and is somatic.

For indels—Quality is based on the QSI_NT field in the INFO column. This score represents the probability that the indel exists and is somatic.

For SNVs and indels, Read Depth is extracted from values listed for DP in the FORMAT column of the cancer sample.

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  Chapter 2  Applying Annotations and Classifications

Applying Annotations and

Classifications

Annotate Variants

Create Custom Annotations

Apply Variant Classifications

Edit Variant Classifications

Manage Classifications

Import Classifications

26

29

31

33

35

37

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Annotate Variants

From the Annotations and Classification tab, use commands on the Annotate menu to annotate variants in the current sample with options to annotate all variants or only those variants specified. All coordinates used in BaseSpace VariantStudio are genomic coordinates on the positive strand.

Always annotate variants before applying filters.

NOTE

An internet connection is required to annotate variants. After annotating, an internet connection is not necessary.

Figure 13 Annotate Menu

Command

Annotate

Custom Annotation

Custom Gene

Annotation

Description

Annotates variants in the project using the following options:

All Variants of Current Sample—Annotates all variants in the current sample. The current sample is listed in the

Current Sample field of the Samples menu.

Exonic Variants of Current Sample—Annotates variants found within an exon plus 20 bp on either side of the exonic region to include the annotation of splice site variants.

Selected Variants of Current Sample—Annotates only the variants that you have selected or filtered.

All Samples—Annotates all variants within each sample imported into the project. This process can take time to complete depending on the number of samples in the project.

Opens a window to browse to the location of the custom annotations file for variant-level annotation. For more information, see

Input File for Custom Variant Annotations on page 29 .

Opens a window to browse to the location of the custom annotations file for gene-level annotation. For more information, see

Input File for Custom Gene Annotations on page 29 .

Opens a window that lists the default transcript for each gene and options for changing to other than the default transcript.

Set Default

Transcripts

NOTE

Although mitochondrial variants can be imported, the annotation database does not provide annotations for these variants.

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Annotation Options

From the Annotate menu, click Annotation Options. The Annotation Options form opens with options to annotate only certain variants.

Figure 14 Variant Option Form

Option

Transcript Annotation

Transcript Source

Type

Forget BaseSpace

Logon

Description

Provides options to annotate only variants in the canonical transcript, which is the longest translated transcript in the gene, and variants in intronic regions.

Annotates variants identified in a specific annotation source, which is RefSeq, by default.

The default can be changed to Ensembl by editing the mode entry in the BaseSpace VariantStudio configuration file

(VariantStudio.exe.config), as follows:

<add key="Mode" value="Ensembl"/>

Close and reopen BaseSpace VariantStudio to enable the change.

Clears BaseSpace login information, such as ID and password.

Set Default Transcripts

1 Click Set Default Transcripts. A window opens that lists the default transcript for each gene. By default, BaseSpace VariantStudio lists the canonical transcript, which is the longest translated transcript in the gene.

2 For genes with multiple transcripts, use the drop-down list to set the default to another transcript.

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Figure 15 Set Default Transcripts

28

Alternatively, click Browse to navigate to a tab-delimited text file containing your preferred default transcripts and click Load.

Input File for Default Transcripts

The input file for default transcripts requires two columns: Gene_Name and Transcript_

Name, as shown in the following example.

Gene_Name

ACTN3

ADH1B

AKAP10

Transcript_Name

NM_003793.3

NM_000668.4

NM_007202.3

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Create Custom Annotations

Applying custom annotations requires a tab-delimited input file with a *.txt extension. One input file is required for variant-level annotations and one for gene-level annotations.

NOTE

BaseSpace VariantStudio assumes that all annotations are expressed in the genomic coordinates on the positive strand, including any custom annotations that are imported into

BaseSpace VariantStudio.

Input File for Custom Variant Annotations

The input file for custom variant annotations requires five columns: Chr, Position, Ref,

Variant, and Annotation, as shown in the following example.

1

1

Chr

1

Position

11046855

11046909

14096821

Ref

G

A

T

Variant

T

T

C

Annotation

Good

Bad

Confirmed

} Chr—The chromosome for the variant (1–22, X, Y, or M).

} Position—The genomic coordinate of the variant on the chromosome (1-based).

}

Ref—The reference base, or bases for an insert or deletion, at the specified position.

}

Variant—The base, or bases for an insert or deletion, at the specified position.

} Annotation—The value assigned to a variant with matching values for chr, position, ref, and variant.

}

(Optional)  Three additional annotation columns are recognized input for custom variant annotations. Use the headings Annotation2, Annotation3, and Annotation4.

NOTE

The Ref field and Variant field must be expressed in VCF format, where indels contain the preceding base in common between the reference and variant allele. For more information, see www.1000genomes.org/wiki/Analysis/Variant Call Format/vcf-variant-call-formatversion-41. On this site, go to step 3, Data Lines, Fixed Fields, and then step 4 Ref.

Input File for Custom Gene Annotations

The input file for gene annotations requires two columns: Gene and Annotation, as shown in the following example.

Gene

AGRN

CCDC39

DHTKD1

Annotation

Myasthenia, limb-girdle, familial

Ciliary dyskinesia, primary, 14

2-aminoadipic 2-oxoadipic aciduria

} Gene—The gene symbol.

}

Annotation—The value assigned to the specified gene.

Apply Custom Annotations

1 Create a custom annotations file using a text editor, such as Notepad, and save it with a *.txt file extension.

2 From the Annotate menu, click Custom Annotations to apply annotations to variants or Custom Gene Annotations to apply annotations to genes.

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3 Browse to the custom annotations file and click OK. This step links the custom annotations file to the project.

4 Use the custom filters in the Filters pane to filter data based on custom annotations. For more information, see

Custom Filters on page 47

.

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Apply Variant Classifications

Introduced in BaseSpace VariantStudio v2.1, you can apply classifications to variants according to their biological impact. Classifications are stored in the classification database.

Figure 16 Classification Menu

Command

Apply Classifications from Database

View Classification

Database

Classification Settings

Description

Use this command to apply classifications to any variants in the current sample that are listed in the classification database. For more information, see

Apply Classifications from Database on page 32 .

Use this command to open the classification database, edit entries in the database, or import classifications from an external file. For more information, see

View Classifications Database on page 35 .

BaseSpace VariantStudio provides five classifications: Benign,

Presumed Benign, Presumed Pathogenic, Pathogenic, and

Unknown Significance. Use this command to add or remove classification categories. For more information, see

Add or Remove

Classification Categories on page 35

.

Variant classifications can be changed at any time in the classification database or changed locally in the current project without changing database entries. For more information, see

Edit Variant Classifications on page 33

.

By default, the classification database is saved locally for use with any BaseSpace

VariantStudio project that is opened locally. For more information, see

Classification

Database Location on page 36 .

A backup of the classification database is created with the first change of each day. For more information, see

Classification Database Backup on page 36

.

For each classified variant, two text fields are available for recording comments about the variant, the Notes field and the Report Fragment field:

} Notes—Information in Notes field is stored in the classification database only.

}

Report Fragment—Information in the Report Fragment field is stored in the classification database and exported as a column in the sample report. For more information, see

Sample Report Overview on page 59

.

There are three ways to apply classifications to variants in a project:

}

From the menu, apply classifications to variants in the current sample that are listed in the classification database.

}

From the Variants table, apply a classification to a selected variant in the Variants table and save the classification to the database.

} From the Variants table, apply a classification to multiple selected variants in the

Variants table and save the classification to the database. The same classification must apply to all selected variants.

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Apply Classifications from Database

Click Apply Classifications from Database. Any variants in the current project that have matching criteria in the classification database are annotated with the classification as specified in the database.

Apply Classifications in the Variants Table

1 Click the icon in the Classification column for the variant you want to classify. The

Classification for Variant in Database window opens, which shows information for the variant and provides a drop-down list of available classification categories.

Figure 17 Classification for Variant in Database

2 Select a classification category from the Classification drop-down list, and enter any applicable comments in the Notes field and Report Fragment field.

3 Click Save Changes to Database. The classification can later be removed from the classification database.

Apply Classifications to Multiple Variants

1 Use shift-click or ctrl-click to select more than one row in the Variants table.

2 Right-click in the Classifications column over a selected row, and then select Classify

Selected Variants.

3 From the Classify Selected Variants window, use the drop-down list to assign a classification. Enter any applicable comments in the Notes field and Report Fragment field.

4 Click OK. The classification assignments are saved to the database automatically.

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Edit Variant Classifications

There are two ways to edit classifications for variants with assigned classifications:

}

Edit variant classifications in the database.

}

Edit classifications locally in the current project without changing database entries.

Edit Classifications in the Database

1 Click the icon in the Classification column for the variant you want to change. A window opens that shows information about the variant, the current classification, and any comments in the Report Fragment field.

2 Click Edit Classification in Classification Database. The Classification for Variant in

Database window opens.

Figure 18 Edit Classifications

3 Do one of the following:

• To change the classification in the database, select a new classification from the

Classification drop-down list and click Save Changes in Database.

• To remove the classification from the database, click Remove Classification from

Database.

Figure 19 Remove Classification from Database

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Edit Variant Classifications Locally

To change variant classifications locally, the variant must already have a classification assigned in the classification database.

1 Click the icon in the Classification column for the variant that you want to edit locally. A window opens that shows information about the variant, the current classification, and any comments in the Report Fragment field.

Figure 20 Reapply Classifications from Database

2 Select a different classification category from the Classification drop-down list.

3 Click OK. The classification is applied to the variant in the current project only. The variant classification recorded in the database can be reapplied to the variant later.

4 To revert the classification to what is assigned in the database, click the

Classification column.

icon in the

5 Click Apply Classification from Classification Database. The classification recorded in the database appears in the Classification field. Click OK.

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Manage Classifications

From the Annotations and Classification tab, use commands on the Classifications menu to view the classification database and manage classification settings.

View Classifications Database

Click View Classification Database to view the entries in the classification database. From this window, you can edit an entry, delete an entry, or import classifications from an internal file.

}

Edit an entry—Select a row, or use shift-click or ctrl-click to select multiple rows. Click

Edit Selected. Reassign a classification or add comments.

} Delete an entry—Select a row, or use shift-click or ctrl-click to select multiple rows.

Click Delete Selected. The entry is permanently deleted from the database.

}

Import classifications—Click Import Classifications and browse to the location of your external classifications file. For more information, see

Import Classifications on page 37

.

Figure 21 View Classification Database

Add or Remove Classification Categories

Click Classification Settings to add, remove, or rename classification categories.

}

Add—In the Add Category field, enter a new category name. Click Add.

}

Remove—Select a category from the list. Click Remove.

} Rename—Select a category from the list. Click Rename and enter a new name.

Figure 22 Classifications Options Dialog Box

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Classification Database Location

By default, the classification database is saved locally in

C:\ProgramData\Illumina\Illumina VariantStudio\ClassificationDb.bin

.

When the database is stored locally, classifications are available for the current project and any future projects opened on that computer.

If the classification database is stored on a network location, classifications are available to projects opened in any installation of BaseSpace VariantStudio with access to that network location.

1 To change the default setting, open the BaseSpace VariantStudio configuration file in

C:\Program Files\Illumina\Illumina VariantStudio\VariantStudio.exe.config

.

2 In the value field of the ClassificationDatabaseFilePath key, enter the preferred network path.

3 Save and close the configuration file.

4 Close and reopen BaseSpace VariantStudio to enable the change.

Classification Database Backup

A backup of the classification database is created the first time the database is changed on any given day.

The backup is named DDMMYYYY.bin and is stored in the folder DatabaseBackups, which is located in the same folder as the classification database,

C:\ProgramData\Illumina\Illumina VariantStudio\DatabaseBackups .

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Import Classifications

To import classifications to the classification database from an external file, create an input file in a tab-delimited text format (TSV) using a *.tsv file extension.

The input file requires five columns: Chr, Position, Ref, Variant, and Classification, as shown in the following example. Optionally, include a Notes column and a Fragment column.

Chr

1

1

1

Position

11046855

11046868

11046909

Ref

G

C

A

Variant

T

T

Classification

Classification 1

Classification 2

Classification 3

Notes

Note 1

Note 2

Note 3

} Chr—The chromosome for the variant (1–22, X, Y, or M).

}

Position—The genomic coordinate of the variant on the chromosome.

}

Ref—The reference base, or bases, for an insert or deletion at the specified position.

} Variant—The base, or bases, for an insert or deletion at the specified position.

} Classification—The value assigned to a variant with matching values for chr, position, ref, and variant. The classification name must match one of the classifications listed in your database.

}

Notes—Note about the entry. Information in this field is not included in the sample report.

} Fragment—Notes about the entry that are intended for the sample report.

NOTE

Make sure that you add any new classification names to the database using the

Classifications Settings command.

Best Practices for Importing Classifications

NOTE

All coordinates used in BaseSpace VariantStudio are genomic coordinates on the positive strand.

Before importing previously classified variants into the BaseSpace VariantStudio classification database, convert classifications to genomic coordinates. This step is especially important for variants that were classified based on HGVSc notations and transcripts.

Importing classifications before converting to genomic coordinates can result in some variants not being annotated with imported classifications when you use the command

Apply Classifications from Database. Because BaseSpace VariantStudio assumes that all annotations are expressed in genomic coordinates on the positive strand, the classification database requires an exact match for variants to be annotated with stored classifications.

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  Chapter 3  Applying Filters

Applying Filters

Apply Filters

Family-Based Filtering Workflows

Create Favorite Filters

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Apply Filters

The Filters pane provides options for applying any combination of filters to the data in your project. Filters are grouped in nine expandable sections: General, Variant, Gene,

Consequence, Population Frequency, Cross Sample Subtraction, Family Based, Custom, and

Classification.

Figure 23 Filters Pane

1 Click the down arrow icon to expand a filter section.

2 From the available options, select filter settings. Use any combination of settings from any number of filters.

3 Click Apply Filters. Filters are applied to the current sample only, not to all samples that are imported into the project.

4 Click Clear Filters to remove applied filters.

NOTE

You can create a filter using any combination of the filter options in the Filters pane, and then save the combination as a single filter. Saved filters can later be applied to other samples. For more information, see

Create Favorite Filters on page 55 .

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General Filters

Use the General filters to filter data by genotype, variant type, and chromosome.

Filter Name

Genotype

Variant Type

Chromosome

Advanced

Setting Description

Filters data to show any combination of heterozygote, homozygote, or hemizygote.

All options are selected by default.

Filters data to show any combination of SNVs, Insertions,

Deletions, or Reference calls.

Filters data to show all chromosomes (default), autosomal chromosomes, or a specific chromosome number.

Filters data based on selections that you make in the

Advanced Filter window.

Advanced Filter Options

Use the Advanced filter options to create a multi-branched Boolean expression for filtering data in the Variants table. As you build the advanced filter, a diagram appears to illustrate the filter and branches in the expression.

1 Select the checkbox labeled Use Advanced Filter and then click Edit Filter. The Create

Advanced Filter window opens.

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Figure 24 Create Advanced Filter Window

2 Select a column heading from the list on the left-hand side.

3 Select an operation from the Operation list.

4 Select either Constant or Parameter.

• To filter on a constant, enter a constant associated with the selection from the lefthand column.

• To filter on a parameter, select a column heading from the list on the right-hand side.

5 Click the generate filter button. A diagram of the filters appears.

6 To add another branch to the advanced filter, select the radio button for either and, or,

xor (exclusive). Then, click Add. A new branch is added to the diagram.

7 Continue selecting options and operators until you have completed the filter.

8 When the advanced filter is complete, click OK.

9 From the Filters pane, click Apply Filters.

Variant Filters

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Use the Variant filters to filter by variant call attributes, variant positions, and variants with specific annotation.

Filter Name

Variant Call

Show only variants

Only variants with

Setting Description

Filters data based on a specified value for variant call quality: pass filter, quality score, read depth, or percentage of variant frequency for the minor allele.

Select the checkbox, and then use the up/down arrows to specify a minimum threshold.

Filters data based on variant position. Options include inside genes and in conserved regions.

Filters data based on the source of annotation. Options include variants without dbSNP ID, with COSMIC annotation, and with ClinVar annotation.

COSMIC and ClinVar annotation enable two more choices:

• If where matches mutant allele is selected and variant has multiple records, only pass variant if at least one matches.

• If where not matches mutant allele is selected and variant has multiple records, only pass variant if none matches.

Gene Filters

Use the Gene filters to filter data by disease, or include or exclude specific genes.

Filter Name

Disease

Include List

Exclude List

Setting Description

Filters data to show genes associated with the specified disease.

Enter the disease name. This field is not case-sensitive.

Filters data to include specified genes.

To include genes, click the button to open the gene list field next to the Include List options, and enter the gene name. This field is not case-sensitive.

Filters data to exclude specified genes.

To exclude genes, click the button to open the gene list field next to the Exclude List options, and enter the gene name. This field is not case-sensitive.

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Filter Name

Min Variant Alleles

Custom and Optional

Gene Annotation

Setting Description

Filters data to show only variants that overlap genes with the specified number of variant alleles. A homozygous variant counts as two variant alleles, while a heterozygous variant counts as one variant allele.

Filters data to show only genes with as specified custom annotation. You can use Boolean logic (AND and OR) to between the Custom and Optional Gene Annotation filters.

The Optional Gene Annotation filter is based on the optional second annotation column that you can import.

NOTE

If you click Clear Filters, the gene list is also cleared. To save a gene list, create a favorite filter. For more information, see

Create Favorite Filters on page 55

.

Consequence Filters

44

Use the Consequence filters to filter data by variants that alter the coding potential of the transcript.

1 Select the checkbox Show only variants that are.

2 Select the checkbox for each individual consequence setting or click Select All.

Filter Name

Missense

PolyPhen, damaging

SIFT, deleterious

Setting Definition

A single base pair substitution that results in the translation of a different amino acid at that position.

Note: PolyPhen and SIFT report only SNVs.

A prediction of a damaging effect of an amino acid substitution on the function of a human protein based on

PolyPhen.

A prediction of a deleterious effect of an amino acid substitution on the function of a human protein based on

SIFT.

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Filter Name

Frameshift

Stop gained

Stop loss

Initiator codon

In-frame insertion

In-frame deletion

Splice

Setting Definition

An insertion or deletion involving a number of base pairs that are not a multiple of three, which disrupts the triple reading frame.

The gain of a stop codon in the coding sequence.

The loss of a stop codon in the coding sequence.

A codon that acts as a start signal for the synthesis of a protein.

An insertion that does not alter the reading frame as a result of the insertion.

A deletion that does not alter the reading frame as a result of the deletion.

An insertion, deletion, or substitution that occurs in a splice region of the gene. A splice is not in a coding region.

Population Frequency Filters

Use the Population Frequency filters to filter data based on the allele frequency in population studies.

Options include global frequency, American, Asian, African, European, and EVS.

}

American, Asian, African, and European are allele frequency from 1000 Genomes.

}

EVS is allele frequency from the NHLBI exome sequencing project.

1 Select the checkbox and then use the up/down arrows to specify a value expressed as percentage.

2 To set the same value to all populations, use the up/down arrows in the Set all to field.

Click Set All.

Cross Sample Subtraction Filter

If multiple samples are present in the project, use the cross sample filter to exclude variants that are also present in another sample.

1 Select the checkbox Use Cross Sample Subtraction.

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2 From the drop-down list, select a sample in the project. Only one sample can be selected as the cross sample filter.

This filter is helpful when filtering variants present in tumor-normal samples.

Family Based Filter

46

Use the Family Based filter to filter for variants that are consistent with user-specified inheritance mode and provided variant data for available family members. The Family

Based Filter requires input of at least one parent or sibling. This filter is useful in identifying candidate disease causing variants.

1 Select the checkbox Use Family Based Filtering.

2 Using the Type drop-down list, select a type from the following choices:

X-linked Recessive—Variant-level filtering of heterozygous variants in affected females that are not present in the father and hemizygous in affected males.

Autosomal Recessive transmission—Gene-level filtering of different heterozygous variants in the same gene in relatives, or variant-level filtering of the same heterozygous variants in both parents.

De novo mutation—Filters variants not present in the relatives. This filter can also be applied using the cross-sample subtraction filter.

Autosomal Dominant transmission—Variant-level filtering of heterozygous variants that are present in the affected relatives, and not present in the unaffected relatives. This filter requires that you indicate the affected relatives.

3 With the child sample set as current, use the drop-down lists to select at least one parent or sibling. All samples to be used in the family-based filtering must be present in the current project.

For more information, see

Family-Based Filtering Workflows on page 49 .

Best Practices When Using the Family Based Filter

}

When using gVCF files for family-based filtering, variants that were not called in the parents are included if the variants are otherwise consistent with the selected inheritance mode.

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}

If you have a gVCF file, use a gVCF viewer such as the Integrative Genomics Browser

(IGV) to examine the no coverage regions. Check for the presence of a disease gene of interest in samples from the child and other family members. For more information, see www.broadinstitute.org/igv/.

Custom Filters

Custom filters enable filtering based on input provided in the custom annotations input file. For more information, see

Create Custom Annotations on page 29 .

Filter Name

Do not filter on custom annotation

Show variants with annotation

Setting Description

Turns off custom annotations. This setting is on by default.

Show variants without annotation

Show variants that contain

Filters data to show variants that match criteria provided in the custom annotations input file with an assigned annotation value in the annotations column.

Filters data to show variants that match criteria provided in the custom annotations input file without an assigned annotation value in the annotations column.

Filters data to show variants that match criteria provided in the custom annotation input file. Options include annotations from any of the four possible annotation columns.

Classification Filter

Use the classification filter to filter by classifications assigned in the classification database.

Any customized classifications appear in the classification filters list.

1 Select the Filter by classification checkbox.

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2 Select the checkbox next to any number of available classifications.

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Family-Based Filtering Workflows

The mode of inheritance, which is the inheritance pattern of a genetic trait or disorder as passed down through generations, is typically one of the following:

}

Autosomal recessive

}

Autosomal dominant

}

X-linked recessive

} De novo mutation

Disease-causing variants co-exist with the disorder according to the mode of inheritance.

Family-based filtering requires at least two samples, the affected person, also known as the proband, and at least one parent or sibling.

Figure 25 Example: Father, Mother, and Proband

} Affected

Because the proband contains thousands of variants that appear deleterious, filtering is necessary to remove variants that are not disease-causing and identify disease-causing variants.

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X-Linked Recessive Transmission Workflow

} A variant is on the X chromosome

}

The variant is heterozygous (0/1) in the mother

}

The variant is not present in the father

}

The variant is homozygous (1/1) in the affected child

Figure 26 X-Linked Recessive Transmission Logic

50

}

}

}

}

Unaffected reference

Unaffected carrier

Affected

Mutation

X-Linked Recessive Transmission Workflow

Proband

Subtract

All variants not on X

Subtract

All variants that are not heterozygous (0/1) in mother

Subtract

All variants that are homozygous (1/1) in father

Subtract

All variants that are not homozygous (1/1) in affected siblings

Subtract

All variants that are homozygous (1/1) in unaffected siblings

Filtering results: Deleterious variants

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Autosomal Recessive Transmission Workflow

There are two possibilities for recessive transmission.

1 A single gene contains a variant that is:

• Heterozygous (0/1) in the mother

• Heterozygous (0/1) in the father

• Homozygous (1/1) in the affected children

Figure 27 Autosomal Recessive Transmission Logic #1

}

}

}

}

Unaffected reference

Unaffected carrier

Affected

Mutation

Autosomal Recessive Transmission Workflow #1

Proband

Subtract

All variants that are homozygous (1/1) in the father, mother, and unaffected siblings

Include

Homozygous (1/1) variants in the child that are heterozygous (0/1) in the mother and father

Subtract

All variants that are not homozygous (1/1) in affected siblings

Subtract

All variants that are homozygous (1/1) in unaffected siblings

Filtering results: Deleterious variants

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2 A single gene contains:

• One variant that is heterozygous (0/1) in the mother

• The same gene contains a different variant that is heterozygous (0/1) in the father

• Both variants are present in the affected child (0/1 and 0/1)

Figure 28 Autosomal Recessive Transmission Logic #2

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}

}

}

}

Unaffected reference

Unaffected carrier

Affected

Mutation

Autosomal Recessive Transmission Workflow #2

Proband

Subtract

All variants that are homozygous (1/1) in the father, mother, and unaffected siblings

Include

Compound heterozygous (0/1) variants, if at least two variants are in the same gene, at least one variant is heterozygous in the father, and other variants are heterozygous in the mother.

Subtract

All variants that are not compound heterozygous (0/1) in affected siblings

Subtract

All variants that are compound heterozygous (0/1) in unaffected siblings

Filtering results: Deleterious variants

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De Novo Mutation Workflow

}

A variant is present (0/1 or 1/1) in the proband

} The variant is not present (0/0) in either parent or siblings

}

Only one child in the family is affected

Figure 29 De Novo Mutation Logic

}

}

}

}

Unaffected reference

Unaffected carrier

Affected

Mutation

De Novo Mutation Workflow

Proband

Subtract

All variants that are heterozygous (0/1) or homozygous (1/1) in the mother, father, and unaffected siblings

Filtering results: Deleterious variants

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Autosomal Dominant Transmission Workflow

} A variant is heterozygous (0/1) in the affected parent

}

The variant is not present (0/0) in the unaffected parent

}

The variant is heterozygous (0/1) in the affected children

Figure 30 Autosomal Dominant Transmission Logic

}

}

}

}

Unaffected reference

Unaffected carrier

Affected

Mutation

Autosomal Dominant Transmission Workflow

Proband

Subtract

All variants that are heterozygous (0/1) in unaffected parent or siblings

Subtract

All variants that are not heterozygous (0/1) in affected parent or siblings

Filtering results: Deleterious variants

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Create Favorite Filters

To save any combination of filtering options for use with a different sample or for later use in another project, save the filtering options as a favorite filter.

The Filter Favorites menu includes commands to save, apply, modify, and manage saved filters.

Figure 31 Filter Favorites Menu Commands

Command

Current

Manage Filters

Save

Save As

Description

Shows the current filter that is applied and a list of available saved filters. Select a saved favorite filter from the drop-down list to apply it to the current sample.

If you change to another sample, a favorite filter applied to the previous sample is not applied automatically to the next sample.

Opens tools for renaming, duplicating, or deleting saved filters.

Saves changes to the currently applied filter.

Opens a dialog box for naming a favorite filter.

Save a Favorite Filter

1 With any combination of filters specified in the Filters pane, select Apply Filter.

2 Click Save As in the Filter Favorites menu.

3 Enter a name for the new filter. Click OK. When a saved filter is applied, the saved filter name appears in the Current field.

Apply a Favorite Filter

1 To apply a saved filter, expand the Current field drop-down list.

2 Select a filter name from the list. The filter is applied automatically.

3 To change to another saved filter, expand the drop-down list in the Current field, and select a different filter name.

4 Alternatively, click the blank entry at the top of the saved filters list to remove the currently applied filter. The variants table is restored to an unfiltered view.

NOTE

The favorite filter is not automatically applied when you move to another sample in your project. To apply a favorite filter, reselect the favorite filter name from the Current dropdown list.

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Modify a Favorite Filter

1 Select additional filtering options from the Filters pane, and click Apply Filter. An asterisk appears next to the saved filter name, which indicates that changes have been applied while the saved filter was selected.

2 To modify the saved filter with the applied filtering options, click Save in the Filter

Favorites menu. The selected saved filter is modified to include the additional filtering options.

Manage Favorite Filters

To rename, duplicate, or delete favorite filters, use the Manage Favorites feature.

Figure 32 Managing Favorite Filters

1 Click Manage Favorites. Names of saved filters appear on the left panel and a block diagram of the selected filter appears on the right panel. To adjust the view of the block diagram, click anywhere on the right panel and use the scrolling feature on your mouse to zoom in or zoom out.

2 Click Done to apply changes.

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  Chapter 4  Generating Reports

Generating Reports

Introduction

Sample Report Overview

Create a Sample Report Template

Create a Sample Report

Export Text Files and Charts

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60

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Introduction

BaseSpace VariantStudio provides tools to export results from a project to an external report.

Use the commands on the Reports tab to create a sample report, and to export data to text files and graphical representations.

Figure 33 Reports Tab

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Sample Report Overview

The sample report consists of five sections plus a footer and is generated as a PDF file or

RTF file, depending on your preference. A sample report includes the following sections:

}

Lab information—Typically, this section is defined in the template and appears as a header in the sample report.

}

Sample information—This section contains details about the sample and appears as a two-column table in the sample report. The first column contains the field name and the second column contains the value. There are two ways to populate this section of the report:

• Specify field names using the Manage Templates feature and then manually enter the value in the text fields.

• Import sample information from an external text file. The text file must have two tab-delimited or comma-separated columns, one for the field name and one for the field value, and use a *.txt, *.csv, or *.tsv extension.

} Test summary—This section is reserved for a description of the test performed. Set up preferred content and formatting in the template. Add information specific to the report when you create the report.

}

Results—This section lists variants in the open project that have a classification assigned. This section is blank if the project does not contain assigned classifications.

Results are formatted in a four-column table with headings of Gene, Variant,

Classification, and Details. Information in the Details column comes from the Report

Fragment field in the classification database.

Figure 34 Example of a Results Section

} Methodology—This section is reserved for a description of the methodology specific to the report. Set up preferred content and formatting in the template. Add information specific to the report when you create the report.

}

References—This section is reserved for references applicable to the contents of the report.

}

Page footer—Typically, this section is defined in the template. For example, the footer can contain the facility address and contact information, or it can be blank.

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Create a Sample Report Template

Use the Manage Templates feature to create a customized template for sample reports. After templates are created, use Manage Templates to duplicate, edit, rename, or delete templates in the template library.

BaseSpace VariantStudio includes an example report template to help in creating a template. The example report template cannot be edited. Instead, create a copy of the example report template. From this copy, rename the template and customize each section of the template using the template tabs.

1 Click Manage Templates. The Manage Report Templates window opens.

Figure 35 Manage Report Templates Window

60

2 Do one of the following:

• Highlight Example Template in the Report Template field and click Duplicate and enter a template name. Click OK.

• To create a template without using the example template, click New and enter a template name. Click OK.

• To edit an existing template, click to highlight the template name in the Report

Template list. This template is now the active template and ready for editing.

3 For each of the following tabs, enter the information to be included in reports using this template. Use the formatting tools to customize the layout. Information included in the following sections of the template are editable when creating the sample report.

Sample Info tab—Specify the sample fields to include in the report. Each field name generates a row in the sample information table.

Lab Information tab—Enter the lab name and location, or other preferred information for the report header.

Test Summary tab—Enter preferred introductory content to begin this section.

Otherwise, leave this section blank in the template.

Methodology tab—Similar to Test Summary, enter preferred introductory content to begin this section. Otherwise, leave this section blank in the template.

References tab—Similar to Test Summary, enter preferred introductory content to begin this section. Otherwise, leave this section blank in the template.

Page Footer tab—Enter preferred content for the template footer, such as contact information. This information appears at the bottom of each page in the report.

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4 Click the Classifications tab. Drag and drop classification names from the Available

Classification list to the Displayed Classification list. The selected classifications are included in any reports using this template and they appear in the order listed.

Figure 36 Classifications for Reporting

5 Click Save Changes.

6 (Optional) With the template name highlighted in the Report Template list, click Make

Default. The current default is listed in the lower-left corner of the Manage Report

Templates window.

7 Click Done. The Manage Report Templates window closes.

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Create a Sample Report

Before proceeding, consider creating a template using the Manage Templates feature. For more information, see

Create a Sample Report Template on page 60 .

1 From the Reports menu, click Sample Report. The Sample Report window opens.

Figure 37 Sample Report, Sample Info Tab

2 From the Report Template drop-down list, select an appropriate template for the report.

3 On the Sample Info tab, enter information in the fields provided or click Import to browse to the location of the text file containing the information.

4 Enter information for the remaining tabs that are not already populated in the selected template.

5 Click Preview to preview the report before generating it.

6 Click Export to PDF or Export to RTF to generate the report.

To save the report contents without generating the report, click Done.

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Export Text Files and Charts

In addition to sample reports, BaseSpace VariantStudio provides tools for exporting to text files and graphical representations of data.

Export Data Files

Exporting filtered variants and all transcripts for variants generates a tab-separated values file. Exporting filter history generates a comma-separated values (CSV) file. These text file formats are not application-specific and can be opened in any text editor. The ANT file is a binary file that contains

Command

Filtered Variants

(TSV)

Description

Exports filtered variants from the current sample.

For variants that overlap multiple genes, only the transcripts that appear on the interface are exported.

Exports all transcripts for filtered variants in the sample.

All Transcripts for

Variants (TSV)

Filter History (CSV) Exports a report of all filters applied to the project.

Export Charts

From the Charts menu, select a preferred format to export results in a histogram or a pie chart.

Command

Histogram

Pie Chart

Description

Generates a histogram of filtered results from the Variants table.

• Use the Plot drop-down list on the generated histogram

(

Figure 38 ) to represent variant quality values or indel

variant length.

• Use Data Source options to show all variants or only filtered variants.

Generates a pie chart of filtered results from the Variants table.

• Use the Plot drop-down list on the generated pie chart

(

Figure 39 ) to represent percentages of variant call filters,

variant type, genotype, coding regions, or filtered variants.

• Use Data Source options to show all variants or only filtered variants.

From the generated chart, click Copy to Clipboard to transfer the image from the BaseSpace

VariantStudio software to an application that supports images.

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Figure 38 Histogram

Figure 39 Pie Chart

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Appendix A  Annotation Sources

Annotation Sources

Annotation Sources 66

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Annotation Sources

Annotation sources are static in the BaseSpace VariantStudio software. Any changes to source databases, such as ClinVar, do not automatically update the annotation sources connected to BaseSpace VariantStudio.

The BaseSpace VariantStudio software includes the following annotation sources:

}

Variant Effect Predictor (VEP)

}

1000 Genomes Project

}

Catalogue of Somatic Mutations in Cancer (COSMIC)

}

ClinVar

}

National Center for Biotechnology (NCBI)

}

National Heart, Lung, and Blood Institute (NHLBI) Exome Variant Server

}

UCSC

VEP v2.8

}

Source:

• Uses data from the Ensembl infrastructure (release 72) ftp.ncbi.nih.gov/snp/organisms/human_9606/VCF/00-All.vcf.gz

• Ensembl infrastructure pulls information from RefSeq (release 56)

}

Values:

• Positional (specific to the position, not necessarily matching the allele):

— GMAF—Global minor allele frequency www.ncbi.nlm.nih.gov/variation/tools/reporter/docs/faq#gmaf

— GMAF allele

— HGNC of overlapping transcripts (for Ensembl only)

For RefSeq, see

NCBI on page 67 .

• Transcript specific:

— Feature

— Feature Type

— Consequence

— cDNA Position

— CDS Position

— Protein Position

— Amino Acids

— Codons

— Exon

— Intron

— HGNC—Direct from Ensembl for ENST

For RefSeq, same as NCBI.

— Distance

— Canonical

— Sift

— PolyPhen

— ENSP

— Domains

— CCDS—Reported for Ensemble annotations only

— HGVSc

— HGVSp

• Positional—Specific to the position, not necessarily matching the allele:

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— Feature ID

— Feature Type

— Consequence

— Motif Name

— Motif Position

— High Influence Position

— Motif Score Change

— Cell Type

1000 Genomes (April 2012 v3)

} Source:

• ftp.1000genomes.ebi.ac.uk/vol1/ftp/release/20110521/ALL.wgs.phase1_release_

v3.20101123.snps_indels_sv.sites.vcf.gz

} Values:

• Ancestral Allele [AA]

• Global allele Frequency [AF]

• Population allele frequencies:

(www.1000genomes.org/category/frequently-asked-questions/population)

— African [AFR_AF]

— Ad Mixed American [AMR_AF]

— East Asian [ASN]

— European [EUR]

COSMIC (v65)

}

Source:

• ngs.sanger.ac.uk/production/cosmic/*_noLimit.vcf.gz

Additional annotation for COSMIC entries were obtained from ftp.sanger.ac.uk/pub/CGP/cosmic/data_export/CosmicCompleteExport_*.tsv.gz

From the CosmicCompleteExport data file, the following four fields were added:

• primary_site

• site_subtype

• primary_histology

• histology_subtype

ClinVar

}

Version September 5, 2013

} www.ncbi.nlm.nih.gov/clinvar/

NCBI

dbSNP (v137)

}

Source:

• ftp.ncbi.nih.gov/snp/organisms/human_9606/VCF/00-All.vcf.gz

}

Values:

• rsID

RefSeq Transcript ID and HGNC

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} Sources:

• ftp.ncbi.nlm.nih.gov/gene/DATA/gene_info.gz

• ftp.ncbi.nlm.nih.gov/gene/DATA/gene2refseq.gz

} Values:

• Transcript ID and gene name are mapped as follows: Transcript ID > Gene ID >

HGNC, using the NCBI unique Gene ID to link the two databases.

NHLBI Exome Variant Server

}

Version ESP6500SI-V2 (updated June 7, 2013)

}

Source: evs.gs.washington.edu/EVS/

• ESP6500SI-V2-SSA137.dbSNP138.snps_indels.vcf.tar.gz

• ESP6500SI-V2.coverage.all_sites.txt.tar.gz

}

Values:

• For SNVs and indels—Allele frequency computed from the TAC field, which reports alternate alleles observed and reference alleles observed

• From the all_sites file (identifies captured positions, even if a variant is not present)

— TotalSamplesCovered

— AvgSampleReadDepth

UCSC (hg19)

Placental mammalian phastCons elements (downloaded from UCSC table browser)

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Index

1

1000 Genomes

annotation source 67

in variants table 14

A

alelle frequency 14, 45

annotation

commands 26 custom 26

options 27

sources 66

transcript source 27

B

BaseSpace File Browser 6

BED file, importing 10

C

cancer analysis pipeline 22

canonical transcript 27

classification filter 47

classifications

adding, removing 35

applying to variants 31 database 31

database backup 36 database storage options 36

edit database 33

importing 37

in reports 60

in variants table 12

report fragments 31

ClinVar

annotation source 67

filter annotations with 43

in variants table 16

codon, intiatator 44

consequence 14

consequence filters 44

COSMIC

annotation source 67

filter annotations with 43

in variants table 15

cross sample subtraction filter 45

custom annotation 26

applying 29 creating 29

filters 47

in variants table 13

input file, genes 29 input file, variants 29

customer support 71

D

de novo mutation, filtering by 46

BaseSpace VariantStudio v2.2 Software User Guide

deletion, in-frame 44

documentation 71

dominant transmission, filtering by 46

E

Ensembl

annotation source 66

in variants table 14

Entrez Gene ID 16

EVS 45

F

family based filtering 46

favorite filters 55

filters

advanced 41

applying 7

classification 47

clear history 7 clearing 7

consequence 44

creating favorites 55

cross sample substraction 45

custom 47

family based 46

gene 43

general 41

history pane 7

history reporting 63

interface 6

population frequency 45

variants 43

filters pane 7, 40

frameshift 44

G

Gene ID 16

gene list

filtering 43

importing from 10

gene view 8

GeneReviews 17

genes table 16

genome VCF 5

GQX

in variants table 13

in VCF file 21

H

help, technical 71

HGNC

in variants table 14 transcript in variants table 14

HGVS 14

histogram, creating 63

69

70

I

import commands 10 import options 10

initiator codon 44

input file

default transcripts 28

insertion, in-frame 44

installation, side-by-side 3

M

missense 44

N

NCBI 67

NHLBI Exome Variant Server 68

no-call regions table 17

P

padding with import 10

pie chart, creating 63

PolyPhen 14, 44

population frequency 45

PubMed

in genes table 17

R

recessive transmission, filtering by 46

reporting

filter history 63

sample report 59, 62

templates 60

variants 63

reports 58

requirements

system 2

VCF input 4

S sample reports

creating 59, 62

creating a templates 60

SIFT 14, 44

somatic variant calls 22

splice 44

system requirements 2

T

table views, modifying 18

tables

genes 6, 16

no-call regions 6, 17

variants 6, 12

technical assistance 71

templates

example report 60 sample report 60

transcript source 27

transcripts

input file 27

input file, default transcripts 28

setting default 27

U

UCSC 68

V variants

add to current sample 10

annotating 26

importing 10

variants table 12

VCF files

fields in the variants table 21

gVCF 5

import options 10 importing 10

requirements 4

somatic VCF 22

VEP 66

W

workflow 2

X

X-linked variants, filtering by 46

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Technical Assistance

For technical assistance, contact Illumina Technical Support.

Table 1 Illumina General Contact Information

Illumina Website

www.illumina.com

Email

[email protected]

Table 2 Illumina Customer Support Telephone Numbers

Region

North America

Austria

Belgium

Denmark

Finland

France

Germany

Ireland

Contact Number

1.800.809.4566

0800.296575

0800.81102

80882346

0800.918363

0800.911850

0800.180.8994

1.800.812949

Region

Italy

Netherlands

Norway

Spain

Sweden

Switzerland

United Kingdom

Other countries

Contact Number

800.874909

0800.0223859

800.16836

900.812168

020790181

0800.563118

0800.917.0041

+44.1799.534000

Safety Data Sheets

Safety data sheets (SDSs) are available on the Illumina website at support.illumina.com/sds.ilmn.

Product Documentation

Product documentation in PDF is available for download from the Illumina website. Go to support.illumina.com, select a product, then click Documentation & Literature.

BaseSpace VariantStudio v2.2 Software User Guide

71

Illumina

Headquartered in San Diego, California, U.S.A.

+1.800.809.ILMN (4566)

+1.858.202.4566 (outside North America) [email protected]

www.illumina.com

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