Illumina Connected Annotations

Illumina Connected Annotations, also known as Illumina Annotation Engine (IAE) or Nirvana provides translational research-grade annotation of genomic variants (SNVs, MNVs, insertions, deletions, indels, STRs, gene fusions, and SVs (including CNVs). It can be run as a stand-alone package, or integrated into larger software tools that require variant annotation.

Users can annotate VCF files by enabling annotation on the DRAGEN command-line or by running the standalone tool.

The input to Illumina Connected Annotations are VCFs and the output is either a structured JSON representation of all annotation and sample information (as extracted from the VCF) or an annotated VCF. Please note that the annotated VCF only contains a subset of the annotations provided in the JSON output. This tool can handle multi-sample VCFs.

NOTE: Before running Annotations, the external data sources, gene models, and reference genome needs to be downloaded from our annotation server.

By default, the Annotations binaries are located in the <INSTALL_PATH>/share/nirvana directory. This directory includes two files: Nirvana (Illumina Connected Annotations) and DataManager.

Limitations

Illumina Connected Annotations and the Downloader are compatible with the following platforms:

• CentOS 7, Oracle 8 and other modern Linux distributions using x64 processors.

Downloading Annotation Data Files

Variant annotation in Dragen requires additional annotation data files that have to be downloaded prior to running any Dragen pipeline that requires variant annotation. To download, please use the DataManager software that is located in <INSTALL_PATH>/share/nirvana/DataManager. To be able to download data successfully, a credential file has to be created.

A credential file will contain an Illumina API key and a premium data source license that you have obtained. To generate Illumina API key, You have to create an Illumina account via this link. As for the premium data license, you can use your existing license key and secret if you already have one or your Dragen serial number. You can use this command to get your Dragen serial number:

dragen_info -b | grep Serial

An example of credential file with Dragen serial number information:

{
  "MyIlluminaApiKey": "<your Illumina account api key>",
  "DragenSerialNo": "<your Dragen server serial no.>"
}

Please follow the guide here for more detail information to create the credential file including how to create Illumina account and generating MyIlluminaApiKey.

Once the credential file is created with your Illumina API key and your premium data license information, you can start to download those files. Dragen has a predefined set of files that has to be downloaded. The configurations are in Dragen resources directory <INSTALL_PATH>/resources/annotation. There are several JSON config files in the directory:

• File all_annotations_GRCh37.json : configurations for producing full variant annotation (running Dragen with parameter --enable-variant-annotation true) for GRCh37 assembly

• File all_annotations_GRCh38.json : configurations for producing full variant annotation (running Dragen with parameter --enable-variant-annotation true) for GRCh38 assembly

• File germline_tagging_annotations_GRCh37.json : configurations required when running Dragen pipeline that perform germline tagging step for GRCh37 assembly

• File germline_tagging_annotations_GRCh38.json : configurations required when running Dragen pipeline that perform germline tagging step for GRCh38 assembly

• File tmb_annotations_GRCh37.json : configurations required when running Dragen pipeline that perform TMB step for GRCh37 assembly (this file will also contain all data that are defined in germline_tagging_annotations_GRCh37.json)

• File tmb_annotations_GRCh38.json : configurations required when running Dragen pipeline that perform TMB step for GRCh38 assembly (this file will also contain all data that are defined in germline_tagging_annotations_GRCh38.json)

To download, execute the command below for each of the JSON file defined above:

.<INSTALL_PATH>/share/nirvana/DataManager download -r [assembly] --credentials-file [path to credential file] --dir [path to directory for the downloaded data] --versions-config [path to the JSON file in the resources folder]

For the --dir argument you can use the same directory for all config file so that it is stored in the same directory. This directory path will be used for parameter --variant-annotation-data when running Dragen.

NOTE:

• If you have run the DataManager for tmb_annotations_[assembly].json, you don't need to run it for germline_tagging_annotations_[assembly].json.

• Data defined in tmb_annotations_[assembly].json are needed if Dragen is run with --enable-tmb true parameter. Without data defined in tmb_annotations_[assembly].json available, running Dragen will result in error.

• Data defined in germline_tagging_annotations_[assembly].json are needed if Dragen is run with --vc-enable-germline-tagging true parameter. Without data defined in tmb_annotations_[assembly].json available, running Dragen will result in error.

For complete guide on how to use DataManager, please read this guide here.

Below is the output example when running the DataManager to download using all_annotations_GRCh38.json config file:

[INSTALL_PATH]/DataManager download -r GRCh38 --credentials-file [path to credential file] --dir [path to dir] --versions-config [path to resource directory]/all_annotations_GRCh38.json

---------------------------------------------------------------------------
DataManager                                         (c) 2024 Illumina, Inc.
                                                                     3.25.0
---------------------------------------------------------------------------

Listing annotation files in local directory...
Requesting remote file information to be downloaded...
Remote file list received!
Syncing local files with requested files
Requesting remote file information to be downloaded...
Start downloading files.
Downloading file DANN_20200205.gsa...
Downloading file Homo_sapiens.GRCh38.Nirvana.dat...
Downloading file Gerp_20110522.gsa...
Downloading file PrimateAI_0.2.nsa...
Downloading file PromoterAI_1.0.esa...
Downloading file REVEL_20200205.nsa...
Downloading file GRCh38.RefSeq.ndb...
Downloading file TOPMed_freeze_5.nsa...
Downloading file ClinGen_disease_validity_curations_20240910.nga...
Downloading file ClinGen_Dosage_Sensitivity_Map_20240910.nga...
Downloading file ClinGen_Dosage_Sensitivity_Map_20240910.nsi...
Downloading file ClinGen_20160414.nsi...
Downloading file ClinVar_20240902.nsa...
Downloading file ClinVar_20240902.nsi...
Downloading file ClinVarPreview_preview_20240902.nsa...
Downloading file ClinVarPreview_preview_20240902.nsi...
Downloading file Cosmic_Cancer_Gene_Census_99.ega...
Downloading file COSMIC_GeneFusions_99.efj...
Downloading file COSMIC_99.esa...
Downloading file dbSNP_156.nsa...
Downloading file dbSNP_151_globalMinor.nsa...
Downloading file gnomAD_gene_scores_4.1.nga...
Downloading file gnomAD_LCR_2.1.lcr...
Downloading file gnomAD_4.1.nsa...
Downloading file gnomAD_SV_4.1.nsi...
Downloading file gnomAD-cnv_4.1.nsi...
Downloading file gnomAD_exome_4.1.nsa...
Downloading file MITOMAP_20200819.nsa...
Downloading file MITOMAP_SV_20200819.nsi...
Downloading file OMIM_20240910.ega...
Downloading file 1000_Genomes_Project_Phase_3_v3_plus_refMinor.rma...
Downloading file 1000_Genomes_Project_Phase_3_v3_plus.nsa...
Downloading file 1000_Genomes_Project_(SV)_Phase_3_v5a.nsi...
Downloading file phyloP_hg38.npd...
Downloading file PhyloPPrimate_1.0.gsa...
Downloading file PrimateAI-3D_1.0.esa...
Downloading file SpliceAi_1.3.esa...
============= Downloading ==============

SpliceAi_1.3.esa (with index)           : [##################################################] 100%
PhyloPPrimate_1.0.gsa (with index)      : [##################################################] 100%
PrimateAI-3D_1.0.esa (with index)       : [##################################################] 100%
======== Download Completed =========   : [##################################################] 100%
Submitting download usage...
Submitting download usage finished!
Obtaining Data License...


---------------------------------------------------------------------------
                            Download Summary
---------------------------------------------------------------------------
Download success:
Total size: 63.61 GB
Downloaded files:
- DANN_20200205.gsa.idx
- PrimateAI_0.2.nsa.idx
- Gerp_20110522.gsa.idx
- PrimateAI_0.2.nsa
- PromoterAI_1.0.esa.idx
- PromoterAI_1.0.esa
- Homo_sapiens.GRCh38.Nirvana.dat
- REVEL_20200205.nsa.idx
- GRCh38.RefSeq.ndb.idx
- GRCh38.RefSeq.ndb
- REVEL_20200205.nsa
- TOPMed_freeze_5.nsa.idx
- ClinGen_disease_validity_curations_20240910.nga
- ClinGen_Dosage_Sensitivity_Map_20240910.nga
- ClinGen_Dosage_Sensitivity_Map_20240910.nsi
- ClinGen_20160414.nsi
- ClinVar_20240902.nsa.idx
- ClinVar_20240902.nsa
- ClinVar_20240902.nsi
- ClinVarPreview_preview_20240902.nsa.idx
- ClinVarPreview_preview_20240902.nsa
- ClinVarPreview_preview_20240902.nsi
- Cosmic_Cancer_Gene_Census_99.ega
- COSMIC_GeneFusions_99.efj
- COSMIC_99.esa.idx
- TOPMed_freeze_5.nsa
- COSMIC_99.esa
- dbSNP_156.nsa.idx
- dbSNP_151_globalMinor.nsa.idx
- dbSNP_151_globalMinor.nsa
- gnomAD_gene_scores_4.1.nga
- gnomAD_LCR_2.1.lcr
- gnomAD_4.1.nsa.idx
- Gerp_20110522.gsa
- gnomAD_SV_4.1.nsi
- gnomAD-cnv_4.1.nsi
- gnomAD_exome_4.1.nsa.idx
- DANN_20200205.gsa
- MITOMAP_20200819.nsa.idx
- MITOMAP_20200819.nsa
- MITOMAP_SV_20200819.nsi
- OMIM_20240910.ega
- 1000_Genomes_Project_Phase_3_v3_plus_refMinor.rma.idx
- 1000_Genomes_Project_Phase_3_v3_plus_refMinor.rma
- 1000_Genomes_Project_Phase_3_v3_plus.nsa.idx
- 1000_Genomes_Project_Phase_3_v3_plus.nsa
- 1000_Genomes_Project_(SV)_Phase_3_v5a.nsi
- phyloP_hg38.npd.idx
- dbSNP_156.nsa
- PhyloPPrimate_1.0.gsa.idx
- gnomAD_exome_4.1.nsa
- PrimateAI-3D_1.0.esa.idx
- phyloP_hg38.npd
- SpliceAi_1.3.esa.idx
- PrimateAI-3D_1.0.esa
- SpliceAi_1.3.esa
- PhyloPPrimate_1.0.gsa
- gnomAD_4.1.nsa
Data license saved: /home/nirvana_resources/nirvana_bin/premium.lic

Peak memory usage: 2.514 GB
Time: 00:04:08.6

Annotate Files (via DRAGEN command-line)

To automatically annotate output VCFs, please add the following command-line arguments:

Argument	Example	Description
--enable-variant-annotation	true	enables annotation if the pipeline supports it
--variant-annotation-data	/path/to/your/NirvanaData	the location where you downloaded the Nirvana annotation files
--variant-annotation-assembly	GRCh38	the genome assembly - either GRCh37 or GRCh38. To annotate hg19 variants, please use GRCh37
--annotation-data-config	/path/to/resources/annotation/all_annotations_GRCh38.json	path to data config file used for data downloading (same as --versions-config)

All the command-line arguments shown together:

--enable-variant-annotation true --variant-annotation-data /path/to/your/NirvanaData --variant-annotation-assembly GRCh38 --annotation-data-config /path/to/resources/annotation/all_annotations_GRCh38.json

Annotate Files (via standalone Illumina Connected Annotations)

1. Please note that Illumina Connected Annotations supports uncompressed VCF files and bgzip compressed VCF files. VCF files that have been compressed by standard gzip are not supported.

2. To annotate the file, enter the following command:

<INSTALL_PATH>/share/nirvana/Nirvana -c [path to data dir]/Cache \
-r [path to data dir]/References/Homo_sapiens.GRCh38.Nirvana.dat \
--sd [path to data dir]/SupplementaryAnnotation/GRCh38 \
-l [path to credential file] --versions-config [path to resource directory]/all_annotations_GRCh38.json
-i <input_VCF> -o <output_prefix>

The following are the available command line options:

Option	Value	Example	Description
-c	directory	~/Data/Cache/	Cache directory
-r	directory	~/Data/References/Homo_sapiens.GRCh38.Nirvana.dat	Reference directory
--sd	directory	~/Data/SupplementaryAnnotation/GRCh38	Supplementary annotation directory
-i	path	<input_VCF>	Input VCF path
-o	prefix	<output_prefix>	Output path prefix
-l	file path	credentials.json	path to credential file
--versions-config	file path	all_annotations_GRCh38.json	path to versions config file

Using the example above, Annotations generates the following output.

---------------------------------------------------------------------------
Illumina Connected Annotations                      (c) 2024 Illumina, Inc.
                                                                     3.25.0
---------------------------------------------------------------------------

Data Source              Type                     Status    Comments
---------------------------------------------------------------------------
DANN                     Score                    Loaded    20200205
DECIPHER                 StructuralVariant        Loaded    201509
Ensembl                  GeneModels               Loaded    112
FusionCatcher            GeneFusion               Loaded    1.33
GME                      SmallVariant             Loaded    20160618
GenomeAssembly           GenomeAssembly           Loaded    GRCh38.p14
Gerp                     Score                    Loaded    20110522
HGNC                     GeneModels               Loaded    20240603
MultiZ100Way             Protein                  Loaded    20171006
PrimateAI                SmallVariant             Loaded    0.2
PromoterAI               SmallVariant             Loaded    1.0
REVEL                    SmallVariant             Loaded    20200205
RefSeq                   GeneModels               Loaded    GCF_000001405.40-RS_2023_10
TOPMed                   SmallVariant             Loaded    freeze_5
clingen                  Gene                     Loaded    20240910
clingen                  StructuralVariant        Loaded    20240910
clingen (legacy)         StructuralVariant        Loaded    20160414
clinvar                  SmallVariant             Loaded    20240902
clinvar                  StructuralVariant        Loaded    20240902
clinvar-preview          SmallVariant             Loaded    20240902
clinvar-preview          StructuralVariant        Loaded    20240902
cosmic                   Gene                     Loaded    99
cosmic                   GeneFusion               Loaded    99
cosmic                   SmallVariant             Loaded    99
dbSNP                    SmallVariant             Loaded    156
globalAllele             SmallVariant             Loaded    151
gnomad                   Gene                     Loaded    4.1
gnomad                   LowComplexityRegions     Loaded    2.1
gnomad                   SmallVariant             Loaded    4.1
gnomad                   StructuralVariant        Loaded    4.1
gnomad-exome             SmallVariant             Loaded    4.1
mitomap                  SmallVariant             Loaded    20200819
mitomap                  StructuralVariant        Loaded    20200819
omim                     Gene                     Loaded    20240910
oneKg                    RefMinor                 Loaded    Phase 3 v3plus
oneKg                    SmallVariant             Loaded    Phase 3 v3plus
oneKg                    StructuralVariant        Loaded    Phase 3 v5a
phylopScore              ConservationScore        Loaded    hg38
phylopScore              Score                    Loaded    1.0
primateAI-3D             SmallVariant             Loaded    1.0
spliceAI                 SmallVariant             Loaded    1.3
---------------------------------------------------------------------------

Initialization                                         Time     Positions/s
---------------------------------------------------------------------------
Cache                                               00:00:03.6
SA initialization                                   00:00:08.0
VCF Position Scan                                   00:00:00.0      115,442

Reference                             SA Loading    Annotation   Variants/s
---------------------------------------------------------------------------
chr1                                    00:00:00.5  00:00:02.0        4,980

Summary                                                Time         Percent
---------------------------------------------------------------------------
Initialization                                      00:00:11.7       84.9 %
Annotation                                          00:00:02.0       14.4 %
 |--SA Loading                                      00:00:00.5        4.2 %
 |--Output writing                                  00:00:00.3        2.2 %
License Check                                       00:00:00.0        0.0 %
Usage Report                                        00:00:00.0        0.0 %
---------------------------------------------------------------------------
Gross Annotation rate:                                  718.84 positions/sec

Peak memory usage: 5.867 GB
Time: 00:00:13.8

JSON Output File

Annotations produces an output file in JSON format. Please refer to Illumina Connected Annotations JSON for detailed description of the JSON file.

Version History

Annotations binaries have been included with DRAGEN since v3.5. The table below indicates which version of Annotations binaries were included with different DRAGEN releases, and their AI annotation capabilities.

The Annotations binaries distributed with DRAGEN can not be changed. Newer versions of Annotations are backward compatible, and can therefore annotate output files from older DRAGEN releases.

DRAGEN version(s)	Annotations version	AI annotations
4.4	3.25	spliceAI, primateAI3D
4.3	3.23	spliceAI, primateAI3D
3.9, 3.10, 4.0, 4.1, 4.2	3.16.1	spliceAI, primateAI
3.8	3.14	spliceAI, primateAI
3.6, 3.7	3.9.0	spliceAI, primateAI
3.5	3.6.0	spliceAI, primateAI