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Version: 3.23

Custom Annotations

Overview

While the team tries to keep data sources up-to-date, you might want to start incorporate new annotations ahead of our update cycle. Another common use case involves protected health information (PHI). Custom annotations are a mechanism that enables both use cases.

Here are some examples of how our collaborators use custom annotations:

  • associating context from both a sample-level and a sample cohort level with the variant annotations
  • adding content that is licensed (e.g. HGMD) to the variant annotations

At the moment, we have two different custom annotation file formats. One provides additional annotations to variants (both small variants and SVs) while the other caters to gene annotations.

In both cases, the custom annotation file format is a tab-delimited file that is separated into two parts: the header & the data.

The header is where you can customize how you want the data to appear in the JSON file and provide context about the genome assembly and how Illumina Connected Annotations should match the variants.

At Illumina, there are usually many components downstream of Illumina Connected Annotations that have to parse our annotations. If a customer provides a custom annotation, those downstream tools need to understand more about the data such as:

  • data type (e.g. number, boolean, or a string)
  • data category (e.g. is this an allele count, allele number, allele frequency, etc.)
  • associated population (i.e. if this is an allele frequency)

For each custom annotation, Illumina Connected Annotations uses this context to create a JSON schema that can be sent to downstream tools. If a tool knows that this is an allele frequency, it can validate user input to ensure that it's in the range of [0, 1].

Variant File Format

File Format

Illumina Connected Annotations expects plain text (or gzipped text) files. Using tools like Excel can add extra characters that can break parsing. We highly recommend creating and modifying these files with plain text editor like Notepad, Notepad++ or Atom.

Basic Allele Frequency Example

Create the Custom Annotation TSV

Imagine that you want to create a basic allele frequency custom annotation for small variants. If we visualized the tab-delimited file (TSV), it would look something like this:

Col 1Col 2Col 3Col 4Col 5
#title=MyDataSource
#assembly=GRCh38
#matchVariantsBy=allele
#CHROMPOSREFALTallAf
#categories...AlleleFrequency
#descriptions...ALL
#type...number
chr1623603511TGAT0.000006579
chr1668801894GA0.000006569
chr1911107436GA0.00003291

Here's the full TSV file.

Let's go over the header and discuss the contents:

  • title indicates the name of the JSON key
  • assembly indicates that this data is only valid for GRCh38.
  • matchVariantsBy indicates how annotations should be matched and reported. In this case annotations will be matched and reported by allele.
  • categories provides hints to downstream tools on how they might want to treat the data. In this case, we indicate that it's an allele frequency.
  • descriptions are used in special circumstances to provide more context. Even though column 5 is called allAf, it might not be clear to a downstream tool that this means a global allele frequency using all sub-populations. In this case, ALL indicates the intended population.
  • type indicates to downstream tools the data type. Since allele frequencies are numbers, we'll write number in this column.
Reference Base Checking

Illumina Connected Annotations validates all the reference bases in a custom annotation. If a variant or genomic region is specified that has the wrong reference base, an error will be produced.

Sorting

The variants within each chromosome must be sorted by genomic position.

Convert to Illumina Connected Annotations Format

First we need to convert the TSV file to Illumina Connected Annotations's native file format and let's put that file in a new directory called CA:

$ mkdir CA
$ dotnet bin/Release/netcoreapp2.1/SAUtils.dll customvar \
-r Data/References/Homo_sapiens.GRCh38.Nirvana.dat -i MyDataSource.tsv -o CA
---------------------------------------------------------------------------
SAUtils (c) 2020 Illumina, Inc.
Stromberg, Roy, Lajugie, Jiang, Li, and Kang 3.12.0
---------------------------------------------------------------------------

Chromosome 16 completed in 00:00:00.1
Chromosome 19 completed in 00:00:00.0

Time: 00:00:00.2

Annotate with Illumina Connected Annotations

Let's annotate the following VCF (notice that it's one of the variants that we have in our custom annotation):

##fileformat=VCFv4.1
#CHROM POS ID REF ALT QUAL FILTER INFO
16 68801894 . G A . . .

Here's the full VCF file.

Since Illumina Connected Annotations can handle multiple directories with external annotations, all we need to do is specify our new CA directory in addition to the normal Illumina Connected Annotations command-line.

$ dotnet Annotator.dll -c Data/Cache/GRCh38/Both \
-r Data/References/Homo_sapiens.GRCh38.Nirvana.dat \
--sd Data/SupplementaryAnnotation/GRCh38 --sd CA -i TestCA.vcf -o TestCA
---------------------------------------------------------------------------
IlluminaConnectedAnnotations (c) 2020 Illumina, Inc.
Stromberg, Roy, Lajugie, Jiang, Li, and Kang 3.12.0
---------------------------------------------------------------------------

Initialization Time Positions/s
---------------------------------------------------------------------------
Cache 00:00:01.8
SA Position Scan 00:00:00.0 19

Reference Preload Annotation Variants/s
---------------------------------------------------------------------------
chr16 00:00:00.2 00:00:01.3 1

Summary Time Percent
---------------------------------------------------------------------------
Initialization 00:00:01.9 25.5 %
Preload 00:00:00.2 3.3 %
Annotation 00:00:01.3 18.2 %

Time: 00:00:06.3

Investigate the Results

We would expect the following data to show up in our JSON output file:

      "variants": [
{
"vid": "16-68801894-G-A",
"chromosome": "16",
"begin": 68801894,
"end": 68801894,
"refAllele": "G",
"altAllele": "A",
"variantType": "SNV",
"hgvsg": "NC_000016.10:g.68801894G>A",
"phylopScore": 1,
"MyDataSource": {
"refAllele": "G",
"altAllele": "A",
"allAf": 7e-06
},
"clinvar": [

Here's the full JSON file.

Illumina Connected Annotations preserves up to 6 decimal places for allele frequency data.

Categories & Descriptions Example

Create the Custom Annotation TSV

Building on the previous example, we can add other types of annotations like predictions and general notes.

Col 1Col 2Col 3Col 4Col 5Col 6Col 7
#title=MyDataSource
#assembly=GRCh38
#matchVariantsBy=allele
#CHROMPOSREFALTallAfpathogenicitynotes
#categories...AlleleFrequencyPrediction.
#descriptions...ALL..
#type...numberstringstring
chr1623603511TGAT0.000006579P.
chr1668801894GA0.000006569LPSeen in case 123
chr1911107436GA0.00003291..

Here's the full TSV file.

Placeholders

You can use a period to denote an empty value (much in the same way as periods are used in VCF files to signify missing values). While Illumina Connected Annotations also accepts empty columns in the TSV file, we use them in these examples to promote readability.

Let's go over what's new in this example:

  • Column 6 adds a field called pathogenicity which uses the Prediction category. When using this category, Illumina Connected Annotations will validate to make sure that the field contains either the abbreviations (B, LB, VUS, LP, and P) or the long-form equivalents (e.g. benign or pathogenic).
  • Column 7 adds a field called notes and it doesn't have a category or description. We're just going to use it to add some internal notes.

Annotate with Illumina Connected Annotations

Let's use a new VCF file. It includes all the same positions as our custom annotation file, but only the middle variant also matches the alternate allele (allele-specific match):

##fileformat=VCFv4.1
#CHROM POS ID REF ALT QUAL FILTER INFO
16 23603511 . TG T . . .
16 68801894 . G A . . .
19 11107436 . G C . . .

Here's the full VCF file.

Investigate the Results

Because we specified #matchVariantsBy=allele in our custom annotation file, only the middle variant will get an annotation:

      "variants": [
{
"vid": "16-68801894-G-A",
"chromosome": "16",
"begin": 68801894,
"end": 68801894,
"refAllele": "G",
"altAllele": "A",
"variantType": "SNV",
"hgvsg": "NC_000016.10:g.68801894G>A",
"phylopScore": 1,
"MyDataSource": {
"refAllele": "G",
"altAllele": "A",
"allAf": 7e-06,
"pathogenicity": "LP",
"notes": "Seen in case 123"
},
"clinvar": [

Here's the full JSON file.

Using Positional Matches

What would happen if we changed to #matchVariantsBy=position? Two things will happen. First, our positional variants will now match:

      "variants": [
{
"vid": "16-23603511-TG-T",
"chromosome": "16",
"begin": 23603512,
"end": 23603512,
"refAllele": "G",
"altAllele": "-",
"variantType": "deletion",
"hgvsg": "NC_000016.10:g.23603512delG",
"MyDataSource": [
{
"refAllele": "GA",
"altAllele": "-",
"allAf": 7e-06,
"pathogenicity": "P"
}
],
"clinvar": [

In addition, you will now see an extra flag for our allele-specific variant:

      "variants": [
{
"vid": "16-68801894-G-A",
"chromosome": "16",
"begin": 68801894,
"end": 68801894,
"refAllele": "G",
"altAllele": "A",
"variantType": "SNV",
"hgvsg": "NC_000016.10:g.68801894G>A",
"phylopScore": 1,
"MyDataSource": [
{
"refAllele": "G",
"altAllele": "A",
"allAf": 7e-06,
"pathogenicity": "LP",
"notes": "Seen in case 123",
"isAlleleSpecific": true
}
],
"clinvar": [

Genomic Region Example

Create the Custom Annotation TSV

In the previous example, we added a note for the middle variant, but sometimes it's handy to annotate a genomic region. Consider the following example:

Col 1Col 2Col 3Col 4Col 5
#title=MyDataSource
#assembly=GRCh38
#matchVariantsBy=allele
#CHROMPOSREFENDnotes
#categories....
#descriptions....
#type...string
chr1620000000T70000000Lots of false positives in this region

Here's the full TSV file.

Let's go over what's new in this example:

  • Column 5 now has a field called notes. In essence, it looks exactly like column 7 from our previous example.
  • The main difference is that now one of our custom annotation entries is actually a genomic region. Any variant that overlaps with that region will get a custom annotation.

In the previous example we learned about positional matching vs allele-specific matching. For genomic regions, #matchVariantsBy=allele and #matchVariantsBy=position produce the same result.

Annotate with Illumina Connected Annotations

Let's use the same VCF file as our previous example.

Investigate the Results

    {
"chromosome": "16",
"position": 23603511,
"refAllele": "TG",
"altAlleles": [
"T"
],
"cytogeneticBand": "16p12.2",
"MyDataSource": [
{
"start": 20000000,
"end": 70000000,
"notes": "Lots of false positives in this region",
"reciprocalOverlap": 0,
"annotationOverlap": 0
}
],
"variants": [

Here's the full JSON file.

Reciprocal & Annotation Overlap

For all intervals, Illumina Connected Annotations internally calculates two overlaps: a variant overlap and an annotation overlap. Variant overlap is the percentage of the variant's length that is overlapped. Annotation overlap is the percentage of the annotation's length that is overlap.

Reciprocal overlap is the minimum of those two overlaps. Given that the annotation is 50 Mbp and the deletion is one 1 bp, both overlaps will be pretty close to 0.

We will also see this annotation for the other variant on chr16:

    {
"chromosome": "16",
"position": 68801894,
"refAllele": "G",
"altAlleles": [
"A"
],
"cytogeneticBand": "16q22.1",
"MyDataSource": [
{
"start": 20000000,
"end": 70000000,
"notes": "Lots of false positives in this region",
"reciprocalOverlap": 0,
"annotationOverlap": 0
}
],
"variants": [

Genomic Regions for Structural Variants Example

Create the Custom Annotation TSV

Often we use genomic regions to represent other known CNVs and SVs in the genome. In this use case, we usually don't want to match these regions to other small variants. To force Illumina Connected Annotations to match regions only to other SVs, use the #matchVariantsBy=sv option in the header. Here is an example:

Col 1Col 2Col 3Col 4Col 5
#title=MyDataSource
#assembly=GRCh38
#matchVariantsBy=sv
#CHROMPOSREFENDnotes
#categories....
#descriptions....
#type...string
chr1620000000T70000000Lots of false positives in this region

Here's the full TSV file.

Let's go over what's new in this example:

  • The main difference is the header field #matchVariantsBy=sv which indicates that only structural variants that overlap these genomic regions will receive annotations.

Annotate with Illumina Connected Annotations

Let's use a new VCF file. It contains the first variant from the previous file and a structural variant deletion- both of which overlap the given genomic region.

##fileformat=VCFv4.1
#CHROM POS ID REF ALT QUAL FILTER INFO
16 23603511 . TG T . . .
16 68801894 . G <DEL> . . END=73683789;SVTYPE=DEL

Here's the full VCF file.

Investigate the Results

Note that this time, MyDataSource only showed up for the <DEL> and not the deletion 16-23603511-TG-T.

    {
"chromosome": "16",
"position": 23603511,
"refAllele": "TG",
"altAlleles": [
"T"
],
"cytogeneticBand": "16p12.2",
"variants": [
...
...
{
"chromosome": "16",
"position": 68801894,
"svEnd": 73683789,
"refAllele": "G",
"altAlleles": [
"<DEL>"
],
"cytogeneticBand": "16q22.1-q22.3",
"MyDataSource": [
{
"start": 20000000,
"end": 70000000,
"notes": "Lots of false positives in this region",
"reciprocalOverlap": 0.02396,
"annotationOverlap": 0.02396
}
],
"variants": [

Mixing Small Variants and Genomic Regions

Create the Custom Annotation TSV

Previously we looked at examples that either had small variants or genomic regions. Let's create a file that contains both:

Col 1Col 2Col 3Col 4Col 5Col 6
#title=MyDataSource
#assembly=GRCh38
#matchVariantsBy=allele
#CHROMPOSREFALTENDnotes
#categories.....
#descriptions.....
#type....string
chr1623603511TGAT..
chr1668801894GA..
chr1911107436GA..
chr2110510818C.10699435Interval #1
chr2110510818C<DEL>10699435Interval #2
chr2212370388TT[chr22:12370729[.Known false-positive

Here's the full TSV file.

Let's go over what's new in this example:

  • Column 4 now has the REF field. Exception for the case listed below, this is only used by small variants or translocation breakends.
  • Column 5 now has the END field. This is only used by genomic regions.
  • There are two custom annotations on chr21 and the start and end coordinates look the same, so what's different? Interval #2 has a symbolic allele in the ALT column. When this is used in custom annotation, the start position is treated as the padding base (using VCF conventions). When Illumina Connected Annotations matches a variant to interval #2, it will ignore the padding base and consider the start position to be at position 10510819.

Annotate with Illumina Connected Annotations

Let's use a new VCF file to study how matching works for intervals #1 and #2:

##fileformat=VCFv4.1
#CHROM POS ID REF ALT QUAL FILTER INFO
21 10510818 . C <DUP> . . END=10699435;SVTYPE=DUP
22 12370388 . T T[chr22:12370729[ . . SVTYPE=BND

Here's the full VCF file.

The first variant is similar to the custom annotation labelled "interval #2". Position 10510818 is the padding base, so it effectively starts at position 10510819.

Investigate the Results

  "positions": [
{
"chromosome": "21",
"position": 10510818,
"svEnd": 10699435,
"refAllele": "C",
"altAlleles": [
"<DUP>"
],
"cytogeneticBand": "21p11.2",
"MyDataSource": [
{
"start": 10510818,
"end": 10699435,
"notes": "Interval #1",
"reciprocalOverlap": 0.99999,
"annotationOverlap": 0.99999
},
{
"start": 10510819,
"end": 10699435,
"notes": "Interval #2",
"reciprocalOverlap": 1,
"annotationOverlap": 1
}
],

Here's the full JSON file.

As expected, the variant and interval #2 have matching endpoints, therefore there is 100% overlap. Interval #1 technically starts 1 bp earlier, so its overlap 99.9%.

Further down the JSON file, we find the annotated translocation breakend:

      "variants": [
{
"vid": "22-12370388-T-T[chr22:12370729[",
"chromosome": "22",
"begin": 12370388,
"end": 12370388,
"isStructuralVariant": true,
"refAllele": "T",
"altAllele": "T[chr22:12370729[",
"variantType": "translocation_breakend",
"MyDataSource": {
"refAllele": "T",
"altAllele": "T[chr22:12370729[",
"notes": "Known false-positive"
}
}

Gene File Format

Basic Gene Example

Create the Custom Annotation TSV

Previously we looked at examples that either had small variants or genomic regions, however, sometimes we would like to add custom gene annotations. The gene custom annotation file format looks slightly different:

Col 1Col 2Col 3Col 4
#title=MyDataSource
#geneSymbolgeneIdphenotypenotes
#categories...
#descriptions...
#type.stringstring
TP537157Colorectal cancer, hereditary nonpolyposis, type 5.
KRASENSG00000133703Mismatch repair cancer syndromeSeen in cohort 123

Here's the full TSV file.

Let's go over what's in this example:

  • Column 2 has the geneId field. This can be either an Entrez Gene ID or an Ensembl ID.
Gene Symbols

Gene symbols are always in flux and are being updated on a daily basis at the NCBI and at HGNC. Due to this, Illumina Connected Annotations uses the geneId to match genes rather than the gene symbol. However, to make the custom annotation files easier to read, we've included the geneSymbol column as well.

Unknown Gene IDs

When Illumina Connected Annotations parses the gene custom annotation file, it will note any gene IDs that are currently not recognized in Illumina Connected Annotations. In such a case, Illumina Connected Annotations will display an error showing all the unrecognized gene IDs.

Annotate with Illumina Connected Annotations

Let's use a VCF file that contain variants in TP53 and KRAS:

##fileformat=VCFv4.1
#CHROM POS ID REF ALT QUAL FILTER INFO
12 25227255 . A T . . .
17 7675074 . C A . . .

Here's the full VCF file.

Investigate the Results

  "genes": [
{
"name": "KRAS",
"clingenGeneValidity": [
{
"diseaseId": "MONDO_0009026",
"disease": "Costello syndrome",
"classification": "disputed",
"classificationDate": "2018-07-24"
}
],
"clingenDosageSensitivityMap": {
"haploinsufficiency": "no evidence to suggest that dosage sensitivity is associated with clinical phenotype",
"triplosensitivity": "no evidence to suggest that dosage sensitivity is associated with clinical phenotype"
},
"gnomAD": {
"pLi": 0.000788,
"pRec": 0.789,
"pNull": 0.21,
"synZ": 0.336,
"misZ": 2.32,
"loeuf": 1.24
},
"MyDataSource": {
"phenotype": "Mismatch repair cancer syndrome",
"notes": "Seen in cohort 123"
}
},

This is the abbreviated output for KRAS. Here's the full JSON file if you want to see the complete KRAS entry.

Customizing the Header

Title

For the title, you can provide any string that hasn't already been used. The title should be unique.

caution

Make sure that the title does not conflict with other keys in the JSON file.

For small variants, you can't provide a title that conflicts with other keys in the variant object. Some examples of this would be vid, chromosome, transcripts, etc.. The title should also not conflict with other data source keys like clinvar or gnomad.

For structural variants, you can't provide a title that conflicts with other keys in the position object. Some examples of this would be chromosome, svLength, cytogeneticBand, etc. The title should also not conflict with other data source keys like clingen or dgv.

caution

Care should be taken not to annotate using multiple custom annotations that all use the same title.

Genome Assemblies

The following genome assemblies can be specified:

  • GRCh37
  • GRCh38

Matching Criteria

The matching criteria instructs how Illumina Connected Annotations should match a VCF variant to the custom annotation.

The following matching criteria can be specified:

  • allele - use this when you only want allele-specific matches. This is commonly the case when using allele frequency data sources like gnomAD
  • position - use this when you want positional matches. This is commonly used with disease phenotype data sources like ClinVar
  • sv - use this when you want to match to all other overlapping SVs. This use case arose when we were adding custom annotations for baseline copy number intervals along the genome.

Categories

Categories are not used by Illumina Connected Annotations, but are often used by downstream tools. Categories provide hints for how those tools should filter or display the annotation data.

When a category is specified, Illumina Connected Annotations will provide additional validation for those fields. The following table describes each category:

CategoryDescriptionValidation
AlleleCountallele counts for a specific populationSee the supported populations below
AlleleNumberallele numbers for a specific populationSee the supported populations below
AlleleFrequencyallele frequencies for a specific populationSee the supported populations below
PredictionACMG-style pathogenicity classificationsbenign (B)
likely benign (LB)
VUS
likely pathogenic (LP)
pathogenic (P)
Filterfree text that signals downstream tools to add the column to the filterMax 20 characters
Descriptionfree-text descriptionMax 100 characters
Identifierany IDMax 50 characters
HomozygousCountcount of homozygous individuals for a specific populationSee the supported populations below
Scoreany score valueAny double-precision floating point number

Descriptions

Descriptions are used to add more context to the categories. For now, descriptions are mainly used to associate allele counts, numbers, and frequencies with their respective populations.

Populations

The following populations were specified in the HapMap project, 1000 Genomes Project, ExAC, and gnomAD.

Population CodeSuper-population CodeDescription
ACBAFRAfrican Caribbeans in Barbados
AFRAFRAfrican
ALLALLAll populations
AMRAMRAd Mixed American
ASJAshkenazi Jewish
ASWAFRAmericans of African Ancestry in SW USA
BEBSASBengali from Bangladesh
CDXEASChinese Dai in Xishuangbanna, China
CEUEURUtah Residents (CEPH) with Northern and Western European Ancestry
CHBEASHan Chinese in Beijing, China
CHSEASSouthern Han Chinese
CLMAMRColombians from Medellin, Colombia
EASEASEast Asian
ESNAFREsan in Nigeria
EUREUREuropean
FINEURFinnish in Finland
GBREURBritish in England and Scotland
GIHSASGujarati Indian from Houston, Texas
GWDAFRGambian in Western Divisions in the Gambia
IBSEURIberian population in Spain
ITUSASIndian Telugu from the UK
JPTEASJapanese in Tokyo, Japan
KHVEASKinh in Ho Chi Minh City, Vietnam
LWKAFRLuhya in Webuye, Kenya
MAGAFRMandinka in the Gambia
MKKAFRMaasai in Kinyawa, Kenya
MSLAFRMende in Sierra Leone
MXLAMRMexican Ancestry from Los Angeles, USA
NFEEUREuropean (Non-Finnish)
OTHOTHOther
PELAMRPeruvians from Lima, Peru
PJLSASPunjabi from Lahore, Pakistan
PURAMRPuerto Ricans from Puerto Rico
SASSASSouth Asian
STUSASSri Lankan Tamil from the UK
TSIEURToscani in Italia
YRIAFRYoruba in Ibadan, Nigeria

Data Types

Each custom annotation can be one of the following data types:

  • bool - true or false
  • number - any integer or floating-point number
  • string - text
tip

For boolean variables, only keys with a true value will be output to the JSON object.

Using SAUtils

Illumina Connected Annotations includes a tool called SAUtils that converts various data sources into Illumina Connected Annotations's native binary format. The sub-commands customvar and customgene are used to specify a variant file or a gene file respectively.

Convert Variant File

dotnet bin/Release/netcoreapp2.1/SAUtils.dll customvar \
-r Data/References/Homo_sapiens.GRCh37.Nirvana.dat \
-i MyDataSource.tsv \
-o SupplementaryAnnotation
  • the -r argument specifies the compressed reference path
  • the -i argument specifies the input TSV path
  • the -o argument specifies the output directory

Convert Gene File

dotnet bin/Release/netcoreapp2.1/SAUtils.dll customgene \
-r Data/References/Homo_sapiens.GRCh37.Nirvana.dat \
-c Data/Cache \
-i MyDataSource.tsv \
-o SupplementaryAnnotation
  • the -c argument specifies the Illumina Connected Annotations cache path
  • the -i argument specifies the input TSV path
  • the -o argument specifies the output directory