Explify Analysis Pipeline

Description

The Explify Analysis Pipeline offers a dedicated informatics solution for the Illumina Respiratory Pathogen ID/AMR Enrichment Panel Kit (RPIP), Illumina Urinary Pathogen ID/AMR Enrichment Panel Kit (UPIP), and Illumina Viral Surveillance V2 Kit (VSP V2). The application delivers powerful, simple data analysis for simultaneous detection, quantification, and profiling of microorganisms and antimicrobial resistance (AMR) markers.

• RPIP: Targeted enrichment of >280 RNA and DNA respiratory pathogens, including SARS-CoV-2, Influenza viruses, Respiratory syncytial virus, Mycobacterium and Legionella species, and >4000 AMR markers.

• UPIP: Targeted enrichment of >170 genitourinary pathogens, including fastidious, slow-growing, and anaerobic uropathogens, sexually transmitted microorganisms, and >4000 bacterial AMR markers.

• VSP V2: Targeted enrichment for whole-genome sequencing (WGS) of 200 RNA and DNA viruses prioritized as high-risk to public health, zoonotic surveillance, and biotech, and >200 viral AMR markers.

The Explify Analysis Pipeline can also be used to analyze FASTQ/FASTA read files with a set of custom reference sequences.

Command Line Settings

Option	Description
Required Inputs
--enable-explify	Enables the Explify Analysis Pipeline. (Default=false)
--output-file-prefix	Prefix for all output files.
--output-directory	Directory for all output files.
--explify-sample-list	Input sample list .tsv file with sample IDs, FASTQs, etc.
--explify-test-panel-name	"RPIP", "UPIP", "VSPv2", "Custom".
--explify-test-panel-version	Set to test panel version (e.g. "1.0.0").
--explify-ref-db-dir	Path to root directory for Explify Database files.
Optional Inputs
--intermediate-results-dir	Area for temporary files. Size must be greater than size of all FASTQ files multiplied by 3.
--explify-load-db-ram	Option to load database into RAM if not on ramdisk. (Default=false).
--explify-no-read-qc	Option to turn off read QC on FASTQs before analysis. (Default=false).
--explify-internal-control	Option to set internal control from an accepted list. (Default="Enterobacteria phage T7")
--explify-internal-control-concentration	Option to set internal control concentration. (Default=12100000)
--explify-ncpus	Option to set the number of CPUs available for processing.
--explify-sensitivity-threshold	Option to set sensitivity threshold. Range: 0 < Integer < 1000. Only valid for VSPv2. (Default=5).
--explify-custom-ref-fasta	Reference FASTA file. Required for Custom reference DBs.
--explify-custom-ref-bed	Reference BED file. Optional for Custom reference DBs.

Example Command Line

dragen \
  --enable-explify=true \
  --output-file-prefix <PREFIX> \
  --explify-sample-list /path/to/sample/list/tsv \
  --explify-test-panel-name <"RPIP"/"UPIP"/"VSPv2"/"Custom"> \
  --explify-test-panel-version <VERSION> \
  --explify-ref-db-dir /path/to/root/db/dir \
  --explify-load-db-ram=true \
  --output-directory <OUTPUT_DIR> \
  --intermediate-results-dir <OUTPUT_DIR> \
  --explify-ncpus=1

Input Details

Sample Input List

Applies to: --explify-sample-list

The sample input list is a column-formatted file with tab separations between the columns (i.e., a .tsv file).

SampleID     BatchID     RunID     ControlFlag     FastQs
MySample     MyBatch     MyRun     POS             /path/to/fastq1.gz     /path/to/fastq2.gz

Notes:

• The SampleID values must be unique.

• BatchID and RunID are to help users track and manage sample analyses. Often the BatchID is used to track libraries that were prepared together, and the RunID is used to track sequencing runs. They can also be left blank.

• The ControlFlag value can be POS, NEG, BLANK, or left empty.

  • POS is used to indicate a positive control sample.

  • NEG is used to indicate a negative control sample.

  • BLANK is used to indicate a blank control sample (e.g. buffer only).

• If there are multiple FASTQ files, they are tab delimited.

• Please be very careful when editing tsv files. Some editors replace tabs with spaces without alerting the user.

Internal Control

Applies to: --explify-internal-control, --explify-internal-control-concentration

The user may specify one of the internal controls listed below. If NONE is specified, the internal control concentration is ignored. These are case-sensitive and must be input exactly as they appear:

• Allobacillus halotolerans

• Armored RNA Quant Internal Process Control

• Enterobacteria phage T7 (This is the default)

• Escherichia virus MS2

• Escherichia virus Qbeta

• Escherichia virus T4

• Imtechella halotolerans

• Phocid alphaherpesvirus 1

• Phocine morbillivirus

• Truepera radiovictrix

• NONE

The internal control concentration is an integer representing the number of copies/mL of sample for the internal control.

Reference Databases

Applies to: --explify-ref-db-dir, --explify-test-panel-name, --explify-test-panel-version, --explify-load-db-ram,--explify-custom-ref-fasta, --explify-custom-ref-bed

An Explify Reference Database is required to run the Explify Analysis Pipeline in DRAGEN. The databases are stored remotely and must be downloaded prior to running an analysis. The database download script provided to facilitate the download is described below.

Directory Setup

Prior to downloading the databases, create a directory that will be dedicated to storing them. It is recommended that the directory be on a disk with at least 150 GB of free space. The path to this directory will be used for the -d parameter when the download script is run in subsequent steps: "explify-databases/" is used in the examples below.

Obtaining the Download Script

Download and management of Explify reference databases is handled by a shell script. The script can be downloaded with the following command:

wget -O explify-dbs.sh https://illumina-explify-databases.s3.us-east-1.amazonaws.com/explify-dbs.sh
chmod +x explify-dbs.sh

Seeing What Databases are Available for Download

The search subcommand can be used to list what databases can be downloaded:

$ ./explify-dbs.sh search -d explify-databases/
4 database(s) found meeting those criteria:
- Custom-1.0.0
- RPIP-6.3.0
- UPIP-8.3.0
- VSPv2-2.3.0

• The -d argument is the base directory used for storage of the databases

• Optionally, when a test panel name is specified with the -p argument, the results will be limited to that panel

• Optionally, setting the -n argument will filter the search to databases that have not already been downloaded

Downloading a Database

The download subcommand is used to download the database files for a test panel:

./explify-dbs.sh download -d explify-databases/ -p UPIP -v 8.3.0 -n 20

• The -d argument is the base directory used for storage of the databases

• The -p argument is the test panel name

• The -v argument is the test panel version

• The -n argument is the number of CPUs that can be used to download the files (defaults to 1)

Additional notes:

• In this example, after the UPIP-8.3.0 files are downloaded, additional required files will be downloaded to a subdirectory named "common"

• After the files are downloaded, their checksums will be automatically checked

• Due to the size of some of the files, this command will take some time. It is best to run it via screen or nohup

Listing Downloaded Databases

The list subcommand is used to view the databases that have already been downloaded:

$ ./explify-dbs.sh list -d explify-databases/

• The -d argument is the base directory used for storage of the databases

• Optionally, when a test panel name is specified with the -p argument, the results will be limited to that panel

Checking Database Integrity

The download subcommand will automatically check the file checksums after download. The check subcommand can also be used on its own to check the files:

$ ./explify-dbs.sh check -d explify-databases/ -p UPIP -v 8.3.0 -n 20

• The -d argument is the base directory used for storage of the databases

• The -p argument is the test panel name

• The -v argument is the test panel version

• The -n argument is the number of CPUs that can be used to download the files (defaults to 1)

Using the Databases with the Explify Analysis Pipeline

Assume the Explify database distributable, when unpacked, has a root directory name of /explify-databases. The database files will be organized in this root directory first by test panel type, then by test panel version:

explify-databases/
    Custom/
        1.0.0/
    RPIP/
        6.3.0/
    UPIP/
        8.3.0/
    VSPv2/
        2.3.0/

To run an analysis with RPIP 6.3.0, for example, the following inputs would be needed:

--explify-ref-db-dir /explify-databases
--explify-test-panel-name RPIP
--explify-test-panel-version 6.3.0

The Explify Analysis Pipeline will use these inputs to navigate to the specified database location, namely /explify-databases/RPIP/6.3.0.

If the databases are stored on a normal file system, it is recommended that you set --explify-load-db-ram=true. This will tell the Explify Analysis Pipeline to load the databases into memory for faster analysis. It is also allowable to store the databases on a RAM disk, which reduces load time over many Explify Analysis Pipeline runs. In this case, it is recommended to set --explify-load-db-ram=false.

Using the Custom Database Option

To use a Custom database, references are supplied through a FASTA file via --explify-custom-ref-fasta and an optional BED file via --explify-custom-ref-bed. Note that you must have downloaded the Custom database and set --explify-test-panel-name to "Custom", and set --explify-test-panel-version to the version you have downloaded. The supplied Custom Explify Reference Database is used by the Explify Analysis Pipeline to filter out host reads.

In the FASTA file, sequence names must be unique and must not contain any spaces. If there is any space in the FASTA header, the part before the first space is assumed to be the sequence name. It is recommended to use the following in sequence names: alphabets, numbers, underscore (_), hyphen (-), parentheses ((,)), and period (.). Otherwise, the sequence names may appear different in the output.

The BED file must be tab-delimited with at least 4 columns:

1. chrom: the sequence name as it appears in the FASTA

2. chromStart: start position (always set to 0)

3. chromEnd: end position (sequence length)

4. genomeName: name of the genome, target, or microorganism the sequence belongs to (e.g. Monkeypox virus clade II)

5. segmentName (optional): the name of the segment or gene (e.g. Segment 4 (HA)). Set to 'Full' if the sequence is the full genome

Sequence names must match between the FASTA and BED file, and the same set of sequences must appear in both files. If there are multiple viruses, their names should be unique. For example, if there are multiple Influenza genomes, they should not be labeled with the same virus name in the 4th column.

The BED file controls how sequences are labeled in the output JSON. If the Custom Reference FASTA includes sequences from multiple segments, it is recommended to provide this BED file so that the segments are included under the results of that microorganism.

Output Details

The output of the Explify Analysis Pipeline is a single report.json file containing sample QC and targeted microorganism and AMR marker detection results written to the specified output dirtectory.

Report.json format

Top-Level Node

The fields in the top-level node of the output JSON provide general metadata and version information.

Field	Description
.accession	Sample identifier.
.deploymentEnvironment	The environment in which the results were produced.
.batchId	Identifier used for a batch of samples prepared in the lab at the same time.
.analysisId	Identifier for the analysis.
.runId	Identifier used for a sequencing run.
.controlFlag	Indicates whether the sample is a control. It is based on the ControlFlag field in the sample .tsv and can be set to “POS”, “NEG”, “BLANK”, or “-”.
.dragenVersion	The DRAGEN release version.
.analysisPipelineVersion	The analysis pipeline version.
.testType	"RPIP", "UPIP", "VSPv2", or "Custom".
.testVersion	Version of the test.
.testName	Name of the test, e.g. "Explify® Respiratory Pathogen ID/AMR Panel (RPIP) - Data Analysis Solution".
.testUse	"For Research Use Only. Not for use in diagnostic procedures".
.reportTime	Time the report was generated.
.warnings	A list of warnings encountered during the analysis.
.errors	A list of errors encountered during the analysis.

.qcReport Node

All of the fields are relative to .qcReport. This section provides information about sampleQc

Field	Description
.sampleQc	Sample QC information.
.sampleQc.totalRawBases	Number of base pairs in sample before read QC processing.
.sampleQc.totalRawReads	Number of reads in sample before read QC processing.
.sampleQc.uniqueReads	Nuber of reads in sample before read QC processing.
.sampleQc.uniqueReadsProportion	Proportion of unique reads in sample before read QC processing.
.sampleQc.preQualityMeanReadLength	Average read length before read QC processing.
.sampleQc.postQualityMeanReadLength	Average read length after read QC processing.
.sampleQc.postQualityReads	Number of reads in sample after read QC processing.
.sampleQc.postQualityReadsProportion	Proportion of post-quality reads in smple relative to total raw reads.
.sampleQc.removedInDehostingReads	Number of host reads in sample removed during dehosting.
.sampleQc.removedInDehostingReadsProportion	Proportion of host reads in sample removed relative to total raw reads.
.sampleQc.entropy	Kmer entropy of reads after read QC processing.
.sampleQc.gContent	Proportion of guanine (G) base calls in reads after read QC processing.
.sampleQc.libraryQScore	Quality score of the library after read QC processing.
.sampleQc.enrichmentFactor	Enrichment factor information (calculation requires detection of an appropriate Internal Control).
.sampleQc.enrichmentFactor.value	Enrichment factor value reflecting how well targeted regions were enriched.
.sampleQc.enrichmentFactor.category	Enrichment factor category: "poor", "fair", "good", or "not calculated".

.qcReport.sampleComposition Node

All of the fields are relative to .qcReport.sampleComposition. This section provides information about the composition of the sample.

Field	Description
.readClassification	Proportion of reads classified to the following groups:
.readClassification.targetedMicrobial	Targeted microbial (non-IC) reference sequences
.readClassification.targetedInternalControl	Targeted IC reference sequences
.readClassification.untargeted	Untargeted reference sequences
.readClassification.ambiguous	More than one pathogen class
.readClassification.unclassified	Could not be classified
.readClassification.lowComplexity	Low complexity sequence
.targetedMicrobial	Proportion of targeted reads classified to the following groups:
.targetedMicrobial.viral	Viral targeted sequences
.targetedMicrobial.bacterial	Bacterial targeted sequences
.targetedMicrobial.fungal	Fungal targeted sequences
.targetedMicrobial.parasitic	Parasitic targeted sequences
.targetedMicrobial.bacterialAmr	Bacterial AMR targeted sequences
.untargeted	Proportion of untargeted reads classified to the following groups:
.untargeted.viral	Viral untargeted sequences
.untargeted.bacterial	Bacterial untargeted sequences
.untargeted.fungal	Fungal untargeted sequences
.untargeted.parasitic	Parasitic untargeted sequences
.untargeted.bacterialAmr	Bacterial AMR untargeted sequences
.untargeted.internalControl	Internal Control (IC) untargeted sequences
.untargeted.human	Human sequences
.viral
.viral.targeted
.viral.untargeted
.viral.untargetedSubcategories
.viral.untargetedSubcategories.panel
.viral.untargetedSubcategories.phage
.viral.untargetedSubcategories.other
.bacterial
.bacterial.targeted
.bacterial.untargeted
.bacterial.untargetedSubcategories
.bacterial.untargetedSubcategories.panel
.bacterial.untargetedSubcategories.ribosomalDna
.bacterial.untargetedSubcategories.plasmid
.bacterial.untargetedSubcategories.other
.fungal
.fungal.targeted
.fungal.untargeted
.fungal.untargetedSubcategories
.fungal.untargetedSubcategories.panel
.fungal.untargetedSubcategories.ribosomalDna
.fungal.untargetedSubcategories.other
.parasitic
.parasitic.targeted
.parasitic.untargeted
.parasitic.untargetedSubcategories
.parasitic.untargetedSubcategories.panel
.parasitic.untargetedSubcategories.ribosomalDna
.parasitic.untargetedSubcategories.other
.human
.human.untargeted
.human.untargetedSubcategories
.human.untargetedSubcategories.ribosomalDna
.human.untargetedSubcategories.codingSequence
.human.untargetedSubcategories.other
.internalControl
.internalControl.targeted
.internalControl.untargeted
.microbialAndInternalControl
.microbialAndInternalControl.targeted
.microbialAndInternalControl.untargeted
.bacterialAmr
.bacterialAmr.targeted
.bacterialAmr.untargeted

.qcReport.internalControls Node

The internalControls object is a list that gives the name and RPKM for the 10 possible IC organisms. See the code block below for an example:

[
    {
        "name": "Allobacillus halotolerans",
        "rpkm": 0
    },
    {
        "name": "Armored RNA Quant Internal Process Control",
        "rpkm": 0
    },
    {
        "name": "Enterobacteria phage T7",
        "rpkm": 180323
    },
    {
        "name": "Escherichia virus MS2",
        "rpkm": 0
    },
    {
        "name": "Escherichia virus Qbeta",
        "rpkm": 0
    },
    {
        "name": "Escherichia virus T4",
        "rpkm": 0
    },
    {
        "name": "Imtechella halotolerans",
        "rpkm": 0
    },
    {
        "name": "Phocid alphaherpesvirus 1",
        "rpkm": 0
    },
    {
        "name": "Phocine morbillivirus",
        "rpkm": 0
    },
    {
        "name": "Truepera radiovictrix",
        "rpkm": 0
    }
]

.userOptions Node

The fields are relative to .userOptions

Field	Description
.quantitativeInternalControlName	The quantitative Internal Control used for microorganism absolute quantification (recommendation: Enterobacteria phage T7)
.quantitativeInternalControlConcentration	The quantitative Internal Control concentration (recommendation: 1.21 x 10^7 copies/mL of sample)
.readQcEnabled	Boolean field that indicates whether read QC (trimming and filtering based on quality and read length) was enabled.
.readClassificationSensitivity	Sensitivity threshold for classifying reads. Determines whether alignment should proceed for a microorganism and/or reference sequence. Only used for VSPv2.

.targetReport.microorganisms Node

The fields are relative to .targetReport.microorganisms. The value of the microorganisms field is an array of objects describing organism detections. The following table describes one microorganisms object.

Field	Description
.class	Microorganism class (viral, bacterial, fungal, parasite)
.name	Name of detected microorganism
.coverage	Proportion of targeted microorganism sequence bases that appear in sequencing reads
.ani	Average nucleotide identity of majority consensus sequence to targeted microorganism reference sequences
.medianDepth	Median depth of reads aligned to targeted microorganism reference sequences, indicating the median number of times each targeted microorganism sequence base appears in sequencing reads
.condensedDepthVector	The depths across the microorganism's targeted reference genes, condensed (if needed) down to 256 items.
.rpkm	Normalized representation of the number of reads aligned to targeted microorganism reference sequences (aligned reads per kilobase of targeted sequence per million reads)
.alignedReadCount	The number of reads that aligned to the organism's target genes.
.kmerReadCount	The number of reads that were assigned to the microorganism's targeted genes with k-mer classification.
.absoluteQuantityRatio	Numerical absolute quantification value
.absoluteQuantityRatioFormatted	Formatted absolute quantification value and units
.phenotypicGroup	Grouping indicating general association with normal flora, colonization, or contamination from the environment or other sources, as well as general association with disease
.associatedAmrMarkers	Information about the detected and predicted AMR markers associated with this bacterium. Only present for bacteria.
.associatedAmrMarkers.applicable	A boolean field that indicates whether the bacterium has one or more AMR markers associated with it in the database.
.associatedAmrMarkers.detected	A list of the detected AMR markers associated with this bacterium. Only present for bacteria.
.associatedAmrMarkers.predicted	A list of the predicted AMR markers associated with this bacterium. Only present for bacteria.
.consensusGenomeSequences	Consensus genome information. Included for RPIP viruses only.
.consensusGenomeSequences.sequence	The consensus genome (or segment) sequence.
.consensusGenomeSequences.referenceAccession	The accession for the reference.
.consensusGenomeSequences.referenceDescription	A description of the reference.
.consensusGenomeSequences.referenceLength	The length of the reference genome.
.consensusGenomeSequences.maximumAlignmentLength	The longest contiguous alignment between consensus and reference sequences.
.consensusGenomeSequences.maximumGapLength	The longest contiguous gap (insertion or deletion) within the alignment between consensus and reference sequences.
.consensusGenomeSequences.maximumUnalignedLength	The longest section of the reference sequence not aligned to by the consensus sequence.
.consensusGenomeSequences.coverage	Proportion of reference sequence bases that appear in sequencing reads
.consensusGenomeSequences.ani	Average nucleotide identity of majority consensus sequence to genome reference sequences
.consensusGenomeSequences.alignedReadCount	The number of reads that aligned to the organism's target genes.
.consensusGenomeSequences.medianDepth	Median depth of reads aligned to genome reference sequences, indicating the median number of times each genome sequence base appears in sequencing reads
.consensusGenomeSequences.targetAnnotation	A list of target annotations for the genome. Each annotation is a JSON object with the following fields: start (int), end (int), strand (string), target_name (string), type (string).
.consensusGenomeSequences.condensedDpethVector	The depth vector for the genome, condensed to 256 items.
.consensusTargetSequences	Information about the consensus sequences for the target sequences.
.consensusTargetSequences.sequence	The consensus sequence for the target.
.consensusTargetSequences.name	The name of the target sequence.
.consensusTargetSequences.referenceAccession	The accession of the reference target.
.consensusTargetSequences.depthVector	The full depth vector for this target gene.
.predictionInformation	Information about Explify's automated interpretation results.
.predictionInformation.predictedPresent	Whether Explify interpretation predicts that the organism is present (true/false)
.predictionInformation.notes	List of notes about the interpretation result.
.predictionInformation.subpanels	A list of the subpanels that the organism belongs to.
.predictionInformation.relatedMicroorganisms	An object that gives key metrics for closely-related on- and off-panel organisms that were detected. See below for details.

.targetReport.microorganisms.relatedMicroorganisms Node

The relatedMicroorganisms object includes a list of the organisms that were considered as part of this organism's interpretation. The fields below describe an object in the relatedOrganisms array.

Field	Description
.name	Related microorganism's name
.onPanel	Whether the related microorganism is on the panel or not.
.kmerReadCount	The number of reads assigned to the microorganism using a k-mer based appraoch. This field is only present when this approach is applied. Currently, it is present for UPIP but not RPIP.
.coverage	The coverage to the microorganism resulting from alignment.
.ani	The ANI to the microorganism resulting from alignment.
.alignedReadCount	The read count to the organism resulting from alignment.

.targetReport.microorganisms.variants Node

The fields are relative to .targetReport.microorganisms.variants. The variants object is only present for select viruses.

Field	Description
.referenceAccession	NCBI accession of reference sequence used for variant calling.
.segment	(Influenza A only). Segment number of reference sequence
.ntChange	Nucleotide change associated with the variant
.referencePosition	Variant position in reference sequence
.referenceAllele	Reference allele at same position as the variant
.variantAllele	Variant allele
.depth	Variant depth, indicating the number of times the variant appears in sequencing reads.
.alleleFrequency	Frequency of the variant allele in the sequencing reads.

.targetReport.amrMarkers Node

The fields are relative to .targetReport.amrMarkers. This section provides information about the detected bacterial AMR markers.

Field	Description
.class	Microorganism class (e.g. bacterial)
.cardModelType	AMR marker detection model specified by CARD (homolog, protein variant, rRNA variant)
.cardGeneFamily	AMR marker family name in CARD
.name	AMR marker name
.cardName	Name of marker in the CARD database
.ncbiName	Name of marker in the NCBI database
.referenceAccession	NCBI or CARD accession of AMR marker reference sequence
.coverage	Proportion of reference sequence residues that appear in sequencing reads (protein alignment for homolog and protein variant model types; DNA alignment for rRNA variant model type)
.pid	Percent identity of majority consensus sequence aligned to reference sequence (protein alignment for homolog and protein variant model types; DNA alignment for rRNA variant model type)
.medianDepth	Median depth of reads aligned to AMR marker reference sequence, indicating the median number of times each AMR marker sequence residue appears in sequencing reads (protein alignment for homolog and protein variant model types; DNA alignment for rRNA variant model type)
.rpkm	Median depth of reads aligned to AMR marker reference sequence, indicating the median number of times each AMR marker sequence residue appears in sequencing reads (protein alignment for homolog and protein variant model types; DNA alignment for rRNA variant model type)
.alignedReadCount	The read count to the marker resulting from alignment.
.nucleotideConsensusSequence	(UPIP only) The nucleotide consensus sequence.
.proteinConsensusSequence	(UPIP only) The protein consensus sequence.
.nucleotideDepthVector	The depths across the nucleotide alignment, not condensed.
.proteinDepthVector	The depths across the protein alignment, not condensed.
.associatedMicroorganisms	Lists of the detected and predicted organisms associated with this marker.
.associatedMicroorganisms.all	A list of all organisms associated with this marker.
.associatedMicroorganisms.detected	A list of the detected organisms associated with this marker.
.associatedMicroorganisms.predicted	A list of the predicted organisms associated with this marker.
.predictionInformation	Information about Explify's automated interpretation results.
.predictionInformation.predictedPresent	Whether Explify interpretation predicts that the marker is present (true/false)
.predictionInformation.confidence	Whether the AMR marker is predicted with high or medium confidence.
.predictionInformation.notes	List of notes about the interpretation result.

.targetReport.amrMarkers.variants Node

The fields are relative to targetReport.amrMarkers.variants. This section provides information about variants detected on select bacterial AMR markers.

Field	Description
.category	"Bacterial Variant; Known AMR"
.referenceSourceMicroorganism	Microorganism that reference sequence is associated with in NCBI
.comments	Comments about the variant
.product	The protein product of the gene
.ntChange	The nucleotide change
.referencePosition	The position on the reference sequence
.referenceAllele	The reference sequence at the position of the variant
.variantAllele	The variant sequence
.depth	The depth at the variant position
.alleleFrequency	The frequency of the variant allele in the read pileup
.annotation	Type of change (e.g. "Nonsynonymous Variant")
.aaChange	Amino acid change
.epistaticGroups	List of epistatic groups the variant is associated with.

.customReferences Node

Only present and populated for custom reference analyses. When only a fasta file is submitted (no BED file), each customReferences object will be for a single reference. When a BED file is provided, each customReferences object is for a single organism/genome and can be for one or more references. The values in the Field column are relative to targetReport.customReferences.

Field	Description
.alignedReadCount	Number of reads aligned to the reference or organism.
.ani	Average nucleotide identity of majority consensus sequence to genome reference sequences.
.condensedDepthVector	The depths across the consensus sequences, condensed (if needed) down to 256 items.
.consensusSequences	Array of objects with information about each consensus sequence for this reference or organism.
.coverage	Proportion of reference sequence bases that appear in sequencing reads
.medianDepth	Median depth of reads aligned to reference sequences, indicating the median number of times each genome sequence base appears in sequencing reads
.name	Either the name (accession) of the reference or the organism
.pangoLineage	Pango lineage information for SARS-CoV-2. Only present if pangolin is run.
.rpkm	Normalized representation of the number of reads aligned to targeted microorganism reference sequences (aligned reads per kilobase of targeted sequence per million reads)
.variants	Array of objects with information about variants detected in the reference sequences

.customReferences.consensusSequences Node

consensusSequences is an array of objects with each object describing the results for a single reference. When only a fasta file is submitted (no BED file), there will be only one reference in the array. When a BED file is provided, there could be more than one. The values in the Field column are relative to targetReport.customReferences[].consensusSequences[]

Field	Description
.alignedReadCount	Number of reads aligned to the reference or organism.
.ani	Average nucleotide identity of majority consensus sequence to genome reference sequences.
.coverage	Proportion of reference sequence bases that appear in sequencing reads
.depthVector	Depths for each base in the sequence
.maximumAlignmentLength	The longest contiguous alignment between consensus and reference sequences.
.maximumGapLength	The longest contiguous gap between consensus and reference sequences.
.maximumUnalignedLength	Longest stretch of unaligned sequence
.medianDepth	Median depth of reads aligned to reference sequences, indicating the median number of times each genome sequence base appears in sequencing reads
.referenceAccession	Accession of the sequence
.referenceDescription	Description of the sequence
.referenceLength	Length of the reference sequence
.sequence	The consensus sequence

.customReferences.variants Node

Variants is an array of objects with each object describing a single detected variant. The values in the Field column are relative to targetReport.customReferences[].variants[].

Field	Description
.alleleFrequency	Frequency of the variant allele in the sequencing reads.
.depth	The depth at the variant position
.ntChange	The nucleotide change
.referenceAccession	Accession of the associated reference
.referenceAllele	The reference sequence at the position of the variant
.referencePosition	The position on the reference sequence
.variantAllele	The variant sequence