Explify Analysis Pipeline

Description

The Explify Analysis Pipeline offers a dedicated informatics solution with flexible analysis options for the following Illumina Infectious Disease and Microbiology target-capture enrichment panel kits: the Illumina Respiratory Pathogen ID/AMR Enrichment Panel Kit (RPIP), Illumina Urinary Pathogen ID/AMR Enrichment Panel Kit (UPIP), and Illumina Viral Surveillance Panel V2 Kit (VSP V2). The application delivers easy-to-use, powerful secondary analysis of Illumina sequencing data, with workflows for sample QC, viral WGS (whole-genome sequencing), pathogen detection and quantification, and antimicrobial resistance (AMR) marker profiling. It also supports custom reference sequence analysis.

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

• VSP V2: Target-capture 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.

• Custom: Analyze FASTQ/FASTA read files with a custom reference sequence database.

Note that samples enriched using the Illumina Respiratory Virus Oligo Panel/Respiratory Virus Enrichment Kit (RVOP/RVEK) and Viral Surveillance Panel Kit (VSP) can also be analyzed using the Explify Analysis Pipeline and VSPv2 database.

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-6.5.1
- UPIP-8.6.0
- VSPv2-2.7.0

• 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 8.6.0 -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-8.6.0 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 8.6.0 -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/
        6.5.1/
    UPIP/
        8.6.0/
    VSPv2/
        2.7.0/

To run an analysis with RPIP 6.5.1, for example, the following inputs would be needed:

--explify-ref-db-dir /explify-databases
--explify-test-panel-name RPIP
--explify-test-panel-version 6.5.1

The Explify Analysis Pipeline will use these inputs to navigate to the specified database location, namely /explify-databases/RPIP/6.5.1.

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 should 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 only 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 file 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 file includes sequences from multiple segments, it is recommended to provide a 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 ap.json file written to the specified output directory containing general metadata, version information, sample QC, microorganism, and AMR marker results, as well as detailed test information.

ap.json format

Top-Level Node

The top-level section of the output JSON contains general metadata and version information.

Field	Description
.accession	Identifier used for the sample
.deploymentEnvironment	Environment in which the results were produced
.batchId	Identifier used for the batch of samples processed together
.analysisId	Identifier used for the analysis
.runId	Identifier used for the 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	DRAGEN release version
.analysisPipelineVersion	Analysis Pipeline release version
.testType	Type of test panel ("RPIP", "UPIP", "VSPv2", "Custom")
.testVersion	Test panel release version
.testName	Full name of test panel
.testUse	Test use. "For Research Use Only. Not for use in diagnostic procedures"
.reportTime	Date and time the report was generated
.warnings	List of warnings encountered during the analysis
.errors	List of errors encountered during the analysis

.qcReport.sampleQc Node

This section contains information about sample quality control (QC). The fields are relative to .qcReport.sampleQc

Field	Description
.totalRawBases	Number of base pairs in sample before read QC processing
.totalRawReads	Number of reads in sample before read QC processing
.uniqueReads	Number of distinct reads in sample before read QC processing
.uniqueReadsProportion	Proportion of distinct reads in sample before read QC processing
.preQualityMeanReadLength	Average read length before read QC processing
.postQualityMeanReadLength	Average read length after read QC processing
.postQualityReads	Number of reads in sample after read QC processing, inclusive of any duplicate reads
.postQualityReadsProportion	Proportion of post-quality reads in sample relative to total raw reads
.removedInDehostingReads	Number of host reads in sample removed during dehosting (host = human)
.removedInDehostingReadsProportion	Proportion of host reads in sample removed relative to total raw reads (host = human)
.entropy	Shannon entropy of the counts of 5-mers in the reads after read QC processing, which is a measure of randomness
.gContent	Proportion of guanine (G) base calls in reads after read QC processing
.libraryQScore	Quality score of the library after read QC processing

.qcReport.enrichmentFactor Node

This section contains information about the enrichment factor calculation. Detection of an appropriate Internal Control is required. The fields are relative to .qcReport.enrichmentFactor

Field	Description
.value	Enrichment factor value reflecting how well targeted regions were enriched
.category	Enrichment factor category: "poor", "fair", "good", or "not calculated"

.qcReport.sampleComposition Node

This section contains information about the composition of the sample. The fields are relative to .qcReport.sampleComposition

Field	Description
.readClassification	Proportion of post-quality reads classified to the following categories:
.readClassification.targetedMicrobial	Targeted microbial
.readClassification.targetedInternalControl	Targeted Internal Control
.readClassification.untargeted	Untargeted
.readClassification.ambiguous	More than one category
.readClassification.unclassified	No category
.readClassification.lowComplexity	Low complexity
.targetedMicrobial	Proportion of post-quality targeted microbial reads classified to the following sub-categories:
.targetedMicrobial.viral	Viral targeted
.targetedMicrobial.bacterial	Bacterial targeted
.targetedMicrobial.fungal	Fungal targeted
.targetedMicrobial.parasitic	Parasitic targeted
.targetedMicrobial.bacterialAmr	Bacterial AMR targeted
.untargeted	Proportion of post-quality untargeted reads classified to the following sub-categories:
.untargeted.viral	Viral untargeted
.untargeted.bacterial	Bacterial untargeted
.untargeted.fungal	Fungal untargeted
.untargeted.parasitic	Parasitic untargeted
.untargeted.bacterialAmr	Bacterial AMR untargeted
.untargeted.internalControl	Internal Control untargeted
.untargeted.human	Human untargeted
.viral	Proportion of post-quality viral reads classified to the following categories:
.viral.targeted	Viral targeted
.viral.untargeted	Viral untargeted
.viral.untargetedSubcategories	Proportion of post-quality viral untargeted reads classified to the following sub-categories:
.viral.untargetedSubcategories.panel	Viral panel members
.viral.untargetedSubcategories.phage	Viral phage
.viral.untargetedSubcategories.other	Viral other (not a panel member or phage)
.bacterial	Proportion of post-quality bacterial reads classified to the following categories:
.bacterial.targeted	Bacterial targeted
.bacterial.untargeted	Bacterial untargeted
.bacterial.untargetedSubcategories	Proportion of post-quality bacterial untargeted reads classified to the following sub-categories:
.bacterial.untargetedSubcategories.panel	Bacterial panel members
.bacterial.untargetedSubcategories.ribosomalDna	Bacterial ribosomal DNA (16S)
.bacterial.untargetedSubcategories.plasmid	Bacterial plasmids
.bacterial.untargetedSubcategories.other	Bacterial other (not a panel member, ribosomal DNA, or plasmid)
.fungal	Proportion of post-quality fungal reads classified to the following categories:
.fungal.targeted	Fungal targeted
.fungal.untargeted	Fungal untargeted
.fungal.untargetedSubcategories	Proportion of post-quality fungal untargeted reads classified to the following sub-categories:
.fungal.untargetedSubcategories.panel	Fungal panel members
.fungal.untargetedSubcategories.ribosomalDna	Fungal ribosomal DNA (18S)
.fungal.untargetedSubcategories.other	Fungal other (not a panel member or ribosomal DNA)
.parasitic	Proportion of post-quality parasitic reads classified to the following categories:
.parasitic.targeted	Parasitic targeted
.parasitic.untargeted	Parasitic untargeted
.parasitic.untargetedSubcategories	Proportion of post-quality parasitic untargeted reads classified to the following sub-categories:
.parasitic.untargetedSubcategories.panel	Parasitic panel members
.parasitic.untargetedSubcategories.ribosomalDna	Parasitic ribosomal DNA (18S)
.parasitic.untargetedSubcategories.other	Parasitic other (not a panel member or ribosomal DNA)
.human	Proportion of post-quality human reads classified to the following categories:
.human.untargeted	Human untargeted
.human.untargetedSubcategories	Proportion of post-quality human untargeted reads classified to the following sub-categories:
.human.untargetedSubcategories.ribosomalDna	Human ribosomal DNA
.human.untargetedSubcategories.codingSequence	Human coding sequence
.human.untargetedSubcategories.other	Human other (not ribosomal DNA or coding sequence)
.internalControl	Proportion of post-quality Internal Control reads classified to the following categories:
.internalControl.targeted	Internal Control targeted
.internalControl.untargeted	Internal Control untargeted
.microbialAndInternalControl	Proportion of post-quality Microbial and Internal Control reads classified to the following categories:
.microbialAndInternalControl.targeted	Microbial and Internal Control targeted
.microbialAndInternalControl.untargeted	Microbial and Internal Control untargeted
.bacterialAmr	Proportion of post-quality bacterial AMR reads classified to the following categories:
.bacterialAmr.targeted	Bacterial AMR targeted
.bacterialAmr.untargeted	Bacterial AMR untargeted

.qcReport.internalControls Node

This section contains information about internal control detection. The value of the .qcReport.internalControls field is an array of objects containing name and RPKM information for each Internal Control. 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

This section gives information about analysis options specified by the user. The fields are relative to .userOptions

Field	Description
.quantitativeInternalControlName	Quantitative Internal Control used for microorganism absolute quantification (recommendation: Enterobacteria phage T7)
.quantitativeInternalControlConcentration	Quantitative Internal Control concentration (recommendation: 1.21 x 10^7 copies/mL of sample)
.readQcEnabled	Boolean indicating if read QC (trimming and filtering based on quality and read length) is enabled
.readClassificationSensitivity	(VSP V2 only) Sensitivity threshold for classifying reads. Determines whether alignment should proceed for a microorganism and/or reference sequence. Value is an integer with a valid range of 1 to 1000, inclusive
.customPanelFastaFile	(Custom Panel only) Name of the custom reference FASTA file
.customPanelBedFile	(Custom Panel only) Name of the custom reference BED file

.targetReport.microorganisms[] Node

The value of the .targetReport.microorganisms[] field is an array of objects containing information about detected microorganisms. The following table describes one .targetReport.microorganisms[] object. The fields are relative to .targetReport.microorganisms[]

Field	Description
.class	Microorganism class ("viral", "bacterial", "fungal", "parasite")
.name	Name of microorganism
.coverage	Proportion of targeted microorganism reference sequence bases that appear in sample sequencing reads
.ani	Average nucleotide identity of consensus sequence to targeted microorganism reference sequences
.medianDepth	Median depth of sample sequencing reads aligned to targeted microorganism reference sequences, indicating the median number of times each targeted microorganism reference sequence base appears in sample sequencing reads
.condensedDepthVector	Read depth across the targeted microorganism reference sequences, condensed to 256 bins
.rpkm	Normalized representation of the number of sample sequencing reads aligned to targeted microorganism reference sequences (targeted Reads mapped Per Kilobase of targeted sequence per Million quality-filtered reads)
.alignedReadCount	Number of sample sequencing reads that aligned to targeted microorganism reference sequences
.kmerReadCount	(UPIP only) Number of sample sequencing reads classified to targeted microorganism reference sequences
.absoluteQuantityRatio	Numerical absolute quantification value. Quantitative internal control required for calculation
.absoluteQuantityRatioFormatted	Formatted absolute quantification value with units. Quantitative internal control required for calculation
.phenotypicGroup	(RPIP, UPIP only) Grouping indicating general association with normal flora, colonization, or contamination from the environment or other sources, as well as general association with disease
.associatedAmrMarkers	(Bacteria only) Information about the bacterial AMR markers associated with the microorganism
.associatedAmrMarkers.applicable	Boolean indicating whether one or more bacterial AMR markers are associated with the microorganism
.associatedAmrMarkers.detected	List of detected bacterial AMR markers associated with the microorganism
.associatedAmrMarkers.predicted	List of predicted bacterial AMR markers associated with the microorganism
.consensusGenomeSequences	(RPIP, VSP V2 viruses only) Information about the majority consensus genome (or segment) sequence
.consensusGenomeSequences.sequence	Consensus genome (or segment) sequence bases
.consensusGenomeSequences.referenceAccession	Accession of the reference genome (or segment) sequence
.consensusGenomeSequences.referenceDescription	Description of the reference genome (or segment) sequence
.consensusGenomeSequences.referenceLength	Length of the reference genome (or segment) sequence
.consensusGenomeSequences.maximumAlignmentLength	Longest contiguous alignment between consensus sequence and reference genome (or segment) sequence
.consensusGenomeSequences.maximumGapLength	Longest contiguous alignment gap (insertion or deletion) between consensus sequence and reference genome (or segment) sequence
.consensusGenomeSequences.maximumUnalignedLength	Longest section of the reference genome (or segment) sequence not aligned to by consensus sequence
.consensusGenomeSequences.coverage	Proportion of reference genome (or segment) sequence bases that appear in sample sequencing reads
.consensusGenomeSequences.ani	Average nucleotide identity of consensus sequence to reference genome (or segment) sequence
.consensusGenomeSequences.alignedReadCount	Number of sample sequencing reads that aligned to reference genome (or segment) sequence
.consensusGenomeSequences.medianDepth	Median depth of sample sequencing reads aligned to reference genome (or segment) sequence, indicating the median number of times each reference genome (or segment) sequence base appears in sample sequencing reads
.consensusGenomeSequences.targetAnnotation	List of targeted region annotations for the reference genome (or segment) sequence. Each annotation is a JSON object with the following fields: start (int), end (int), strand (string: "+", "-"), target_name (string), type (string)
.consensusGenomeSequences.condensedDepthVector	Read depth across the reference genome (or segment) sequence, condensed to 256 bins
.consensusTargetSequences	(RPIP viruses only) Information about the majority targeted region consensus sequences
.consensusTargetSequences.sequence	Consensus targeted region sequence bases
.consensusTargetSequences.name	Name of the targeted region
.consensusTargetSequences.referenceAccession	Accession of the targeted region reference sequence
.consensusTargetSequences.depthVector	Read depth across the targeted region reference sequence, not condensed
.predictionInformation	Information about microorganism prediction results
.predictionInformation.predictedPresent	Boolean indicating whether the microorganism passed its reporting logic algorithm
.predictionInformation.notes	List of notes about the prediction result
.predictionInformation.subpanels	List of pre-defined subpanels that the microorganism belongs to
.predictionInformation.relatedMicroorganisms	Array of objects with information about genetically related microorganisms. See below for details
.variants	(all VSP V2 viruses, RPIP: SARS-CoV-2 & FluA/B/C only) Information about viral variants. See below for details

.targetReport.microorganisms[].predictionInformation[].relatedMicroorganisms[] Node

The value of the .targetReport.microorganisms[].predictionInformation[].relatedMicroorganisms[] field is an array of objects containing information about genetically related microorganisms. The following table describes one .targetReport.microorganisms[].predictionInformation[].relatedMicroorganisms[] object. The fields are relative to .targetReport.microorganisms[].predictionInformation[].relatedMicroorganisms[]

Field	Description
.name	Name of related microorganism
.onPanel	Boolean indicating whether the related microorganism is a panel member
.kmerReadCount	(UPIP only) Number of sample sequencing reads classified to related microorganism reference sequences
.coverage	Proportion of related microorganism reference sequence bases that appear in sample sequencing reads
.ani	Average nucleotide identity of consensus sequence to related microorganism reference sequences
.alignedReadCount	Number of sample sequencing reads that aligned to related microorganism reference sequences

.targetReport.microorganisms[].variants[] Node

The value of the .targetReport.microorganisms[].variants[] field is an array of objects containing information about viral variants for all VSP V2 viruses, RPIP: SARS-CoV-2 & FluA/B/C only. The following table describes one .targetReport.microorganisms[].variants[] object. The fields are relative to .targetReport.microorganisms[].variants[]

Field	Description
.referenceAccession	Accession of reference genome (or segment) sequence used for variant calling
.segment	(Segmented viruses only) Segment number of reference segment sequence
.ntChange	Nucleotide change associated with variant
.referencePosition	Variant position in viral reference genome (or segment) sequence
.referenceAllele	Reference allele at variant position
.variantAllele	Variant allele
.depth	Variant depth, indicating the number of times variant position appears in sample sequencing reads
.alleleFrequency	Frequency of variant allele in sample sequencing reads

.targetReport.amrMarkers[] Node

The value of the .targetReport.amrMarkers[] field is an array of objects containing information about detected bacterial AMR markers. The following table describes one .targetReport.amrMarkers[] object. The fields are relative to .targetReport.amrMarkers[]

Field	Description
.class	Microorganism class ("bacterial")
.cardModelType	Bacterial AMR marker model type in the Comprehensive Antibiotic Resistance Database (CARD) ("homolog", "protein variant", "rRNA variant")
.cardGeneFamily	Bacterial AMR marker gene family in the Comprehensive Antibiotic Resistance Database (CARD)
.name	Bacterial AMR marker name
.cardName	Bacterial AMR marker name in the Comprehensive Antibiotic Resistance Database (CARD)
.ncbiName	Bacterial AMR marker name in the National Center for Biotechnology Information (NCBI) Reference Gene Catalog
.referenceAccession	Accession of the bacterial AMR marker reference sequence
.coverage	Proportion of bacterial AMR marker reference sequence residues that appear in sample sequencing reads (protein alignment for "homolog" and "protein variant" model types; nucleotide alignment for "rRNA variant" model type)
.pid	Percent identity of consensus sequence aligned to bacterial AMR marker reference sequence (protein alignment for "homolog" and "protein variant" model types; nucleotide alignment for "rRNA variant" model type)
.medianDepth	Median depth of sample sequencing reads aligned to bacterial AMR marker reference sequence, indicating the median number of times each bacterial AMR marker sequence residue appears in sample sequencing reads (protein alignment for "homolog" and "protein variant" model types; nucleotide alignment for "rRNA variant" model type)
.rpkm	Normalized representation of the number of sample sequencing reads aligned to bacterial AMR reference sequence (protein alignment for "homolog" and "protein variant" model types; nucleotide alignment for "rRNA variant" model type)
.alignedReadCount	Number of sample sequencing reads that aligned to bacterial AMR reference sequence (protein alignment for "homolog" and "protein variant" model types; nucleotide alignment for "rRNA variant" model type)
.nucleotideConsensusSequence	Nucleotide consensus sequence bases
.proteinConsensusSequence	Protein consensus sequence bases
.nucleotideDepthVector	Read depth across the bacterial AMR marker nucleotide reference sequence, not condensed
.proteinDepthVector	Read depth across the bacterial AMR marker protein reference sequence, not condensed
.associatedMicroorganisms	Information about the microorganisms associated with the bacterial AMR marker
.associatedMicroorganisms.all	List of all microorganisms associated with the bacterial AMR marker
.associatedMicroorganisms.detected	List of detected microorganisms associated with the bacterial AMR marker
.associatedMicroorganisms.predicted	List of predicted microorganisms associated with the bacterial AMR marker
.predictionInformation	Information about bacterial AMR marker prediction results
.predictionInformation.predictedPresent	Boolean indicating whether the bacterial AMR marker passed its reporting logic algorithm
.predictionInformation.confidence	Confidence level of bacterial AMR marker prediction ("high", "medium", "low")
.predictionInformation.notes	List of notes about the prediction result

.targetReport.amrMarkers[].variants[] Node

The value of the .targetReport.amrMarkers[].variants[] field is an array of objects containing information about variants for bacterial AMR markers with "protein variant" or "rRNA variant" model types. The following table describes one .targetReport.amrMarkers[].variants[] object. The fields are relative to .targetReport.amrMarkers[].variants[]

Field	Description
.category	Variant category ("Bacterial Variant; Known AMR")
.referenceSourceMicroorganism	Microorganism that reference sequence is associated with in NCBI
.comments	List of additional information regarding the variant
.product	Protein product of gene
.ntChange	Nucleotide change associated with variant
.referencePosition	Variant position in reference sequence
.referenceAllele	Reference allele at variant position
.variantAllele	Variant allele
.depth	Variant depth, indicating the number of times variant position appears in sample sequencing reads
.alleleFrequency	Frequency of variant allele in sample sequencing reads
.annotation	Type of change (e.g. "Nonsynonymous Variant")
.aaChange	Amino acid change associated with variant
.epistaticGroups	List of epistatic groups variant is associated with

.targetReport.customReferences[] Node

This section contains information about custom reference detection results and is only present for custom database analyses. When only a custom reference FASTA file is provided (no BED file), each .targetReport.customReferences[] object contains information for a single reference sequence. When both a FASTA and BED file are provided, each .targetReport.customReferences[] object contains information for a single genome/microorganism, which can be a collection of one or more reference sequences. The fields are relative to .targetReport.customReferences[]

Field	Description
.name	Provided name of custom reference sequence, accession, genome, or microorganism
.coverage	Proportion of custom reference sequence bases that appear in sample sequencing reads
.ani	Average nucleolotide identity of consensus sequence to custom reference sequence or, if specified, collection of one or more custom reference sequences
.medianDepth	Median depth of sample sequencing reads aligned to custom reference sequence or, if specified, collection of one or more custom reference sequences, indicating the med\ian number of times each custom reference sequence base appears in sample sequencing reads
.condensedDepthVector	Read depth across custom reference sequence or, if specified, collection of one or more custom reference sequences, condensed to 256 bins
.rpkm	Normalized number of sample sequencing reads aligned to custom reference sequence or, if specified, collection of one or more custom reference sequences (targeted Reads mapped Per Kilobase of targeted sequence per Million quality-filtered reads)
.alignedReadCount	Number of sample sequencing reads that aligned to custom reference sequence or, if specified, collection of one or more custom reference sequences
.consensusSequences	Array of objects with information about each consensus sequence. See below for details
.variants	Array of objects with information about variants detected in custom reference sequence or, if specified, collection of one or more custom reference sequences. See below for details

.targetReport.customReferences[].consensusSequences[] Node

The value of the .targetReport.customReferences[].consensusSequences[] field is an array of objects containing majority consensus sequence information for a single custom reference sequence. When only a FASTA file is provided (no BED file), there will be only one object in the array. When both a FASTA and BED file are provided, there may be more than one object in the array. The fields are relative to .targetReport.customReferences[].consensusSequences[]

Field	Description
.sequence	Majority consensus sequence bases
.referenceAccession	Accession of custom reference sequence
.referenceDescription	Description of custom reference sequence
.referenceLength	Length of custom reference sequence
.coverage	Proportion of custom reference sequence bases that appear in sample sequencing reads
.ani	Average nucleolotide identity of consensus sequence to custom reference sequence
.medianDepth	Median depth of sample sequencing reads aligned to custom reference sequence, indicating the median number of times each custom reference sequence base appears in sample sequencing reads
.depthVector	Read depth across custom reference sequence, not condensed
.alignedReadCount	Number of sample sequencing reads that aligned to custom reference sequence
.maximumAlignmentLength	Longest contiguous alignment between consensus sequence and custom reference sequence
.maximumGapLength	Longest contiguous alignment gap (insertion or deletion) between consensus sequence and custom reference sequence
.maximumUnalignedLength	Longest section of custom reference sequence not aligned to by consensus sequence

.targetReport.customReferences[].variants[] Node

The value of the .targetReport.customReferences[].variants[] field is an array of objects containing information about a single detected variant. The fields are relative to .targetReport.customReferences[].variants[]

Field	Description
.ntChange	Nucleotide change associated with variant
.referenceAccession	Accession of custom reference sequence used for variant calling
.referencePosition	Variant position in custom reference sequence
.referenceAllele	Reference allele at variant position
.variantAllele	Variant allele
.depth	Variant depth, indicating the number of times variant position appears in sample sequencing reads
.alleleFrequency	Frequency of variant allele in sample sequencing reads