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First, we recommend saving the provided template file with a new name
Do not add any new columns and do not delete any columns from the template file
Do not change or remove any text from the header row. **The "kmer_read_count" metric is only valid with the UPIP enrichment panel.
Rows with microorganism names that are not of interest can be deleted. However, the entire tiered reporting group for certain viruses must be included to preserve tiered reporting logic (if desired). Membership in a tiered reporting group means that a hierarchical relationship is pre-built into the database and the most granular tier level passing reporting thresholds is reported. For example, if Influenza B virus (B/Victoria/2/87-like) or Influenza B virus (B/Yamagata/16/88-like) are reported in a sample then the less granular Influenza B virus reporting name will NOT be reported. See the "Has Tiered Reporting" and "Reporting Tier" columns of the "Microorganisms" table in the Panel Summary for RPIP, RVOP/RVEK, VSP, and VSP V2 to select and see which viruses are reported as part of a tiered reporting group.
Upload the microorganism reporting file to a BaseSpace Project. **It is only necessary to upload the file once.
Select the file by clicking on the "Dataset File(s)" option under the "User-defined specification" section.
See example below for 6 RPIP microorganism reporting names. Prediction logic can be specified on a microorganism-by-microorganism basis using multiple parameters and combinatorial logical expressions.
Acinetobacter baumannii
default
Cryptococcus neoformans
coverage
0.3
Escherichia coli
aligned_read_count
200
Human adenovirus E
(coverage AND median_depth) OR (aligned_read_count AND ani)
0.1
1
0.95
100
Human bocavirus 1 (HBoV1)
rpkm OR (ani AND coverage) OR median_depth
0.2
5
0.9
5
Klebsiella pneumoniae
default AND coverage
0.5
DRAGEN Microbial Enrichment Plus offers a dedicated informatics solution with flexible analysis options for Illumina Infectious Disease and Microbiology target-capture enrichment panel kits. The app 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 user-defined microorganism reporting thresholds and custom reference sequence analysis.
FASTQ files
(all panels except where noted, (*) indicates applicable to custom reference sequence analysis)
Read QC* (optional)
Dehosting* (human read removal)
Sample QC (sample composition and enrichment factor calculations. Internal control required to calculate the enrichment factor) – RPIP, UPIP, VSP V2
Microorganism classification (configurable sensitivity) - RVOP, VSP, VSP V2
Microorganism detection (alignment, consensus generation, variant calling)
Microorganism quantification (quantitative internal control required) – RPIP, UPIP, VSP V2
Microorganism reporting thresholds (proprietary algorithms or user-defined reporting logic)
Bacterial AMR marker analysis (nucleotide and protein alignment, consensus generation, variant calling and annotation) – RPIP, UPIP
Viral AMR marker analysis (variant calling and annotation) – RPIP, RVOP, VSP, VSP V2
Viral clade and lineage prediction (Pangolin, Nextclade) – RPIP, RVOP, VSP, VSP V2
Result filters (user-specified filters applied)
Reporting*
Analysis-level outputs: XLSX, HTML, ZIP
Sample-level outputs: JSON, HTML, FASTA (consensus sequences), VCF (viral variants)
DRAGEN Microbial Enrichment Plus is a secondary analysis tool for research use only. Further interpretation, statistical analysis, and downstream analysis of results may be necessary.
(optional)
(if applicable)
(if applicable)
The includes external control, contrived, and environmental samples prepared using the RPIP, UPIP, RVOP, VSP, and VSP V2 target-capture enrichment kits. Example custom reference sequence FASTA and BED files are also included.
A custom reference FASTA file containing one or more reference sequences is required to run the custom reference sequence analysis. 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. An example custom reference FASTA file is provided in the link below.
To upload a custom reference FASTA file, go to the "Projects" tab and click on the folded paper icon (representing File) to reveal a dropdown menu. Click on "Upload" and select "Files". Within the upload page, select "Other" format for FASTA files, and upload the file as a Biosample. Within the DRAGEN Microbial Enrichment Plus app, under "Custom panel specification" use the "Custom reference FASTA for consensus generation" control to select the uploaded FASTA file.
Optionally, a custom reference BED file may also be provided. 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 grouped and labeled in the output. If the custom reference FASTA file includes sequences from multiple segments of a viral genome, it is recommended to provide a BED file so that the segments are included under the results of that microorganism.
The BED file must be tab-delimited with at least 4 columns:
chrom: the sequence name as it appears in the FASTA
chromStart: start position (always set to 0)
chromEnd: end position (sequence length)
genomeName: name of the genome, target, or microorganism the sequence belongs to (e.g. Monkeypox virus clade II)
segmentName (optional): the name of the segment or gene (e.g. Segment 4 (HA)). Set to 'Full' if the sequence is the full genome
To upload a custom reference BED file, go to the "Projects" tab and click on the folded paper icon (representing File) to reveal a dropdown menu. Click on "Upload" and select "Files". Within the upload page, select "Other" format for BED files, and upload the file as a Biosample. Within the DRAGEN Microbial Enrichment Plus App, under "Custom panel specification" use the "Custom reference BED (optional)" dropdown to select the uploaded BED file.
For Custom Panel analyses, Pangolin is enabled and will run on custom reference sequences with at least 3% coverage that meet these naming conventions:
If only a FASTA file is provided, Pangolin will run on sequences that have a header containing either SARS-CoV-2 or NC_045512
If both a FASTA and BED file are provided, Pangolin will run on sequences where the first column (chrom) contains NC_045512 or the fourth column (genomeName) contains SARS-CoV-2
For Custom Panel analyses, Nextclade is disabled and will not be run. Do not enable Nextclade.
AMR
antimicrobial resistance
CLSI
Clinical and Laboratory Standards Institute
ESBL
extended spectrum beta-lactamase
EUCAST
European Committee on Antimicrobial Susceptibility Testing
mL
milliliter
NGS
next-generation sequencing
RPKM
targeted Reads mapped Per Kilobase of targeted sequence per Million quality-filtered reads
UPIP
Urinary Pathogen ID/AMR Panel
RUO
For Research Use Only. Not for use in diagnostic procedures.
URL
See https://www.illumina.com/ for additional information.
Quantification - when a quantitative Internal Control {ic_name} and concentration {ic_concentration} is specified
UPIP data analysis using DRAGEN Microbial Enrichment Plus detects 35 viruses, 121 bacteria, 14 fungi, 4 parasites, and 4,371 AMR markers, unless filtered reporting options are selected, based on target enriched next-generation sequencing (NGS) of microorganism DNA sequences. Sequencing data are interpreted by the DRAGEN software platform and microorganisms that pass reporting thresholds are reported. Absolute quantification assumes use of {ic_name} as an Internal Control spiked at {ic_concentration} copies/mL of sample. Relative abundance is calculated based on absolute quantities and is expressed as proportion of absolute quantities within each pathogen class (i.e., bacteria, viruses, fungi, parasites). If RPKM for the Internal Control is zero, no absolute quantification is provided, and relative abundance is expressed as proportion of microorganism RPKM values within each pathogen class.
Quantification - when a quantitative Internal Control is NOT specified
UPIP data analysis using DRAGEN Microbial Enrichment Plus detects 35 viruses, 121 bacteria, 14 fungi, 4 parasites, and 4,371 AMR markers, unless filtered reporting options are selected, based on target enriched next-generation sequencing (NGS) of microorganism DNA sequences. Sequencing data are interpreted by the DRAGEN software platform and microorganisms that pass reporting thresholds are reported. Relative abundance is expressed as proportion of microorganism RPKM values within each pathogen class (i.e., bacteria, viruses, fungi, parasites). Internal Control not specified; no absolute quantification provided.
AMR - when "Report bacterial AMR markers only when an associated microorganism is reported" is selected
This test detects 4,371 antimicrobial resistance (AMR) markers and reports associations for 72 microorganisms, 185 antimicrobials, and 33 drug classes, unless filtered reporting options are selected. AMR markers are based on the Comprehensive Antibiotic Research Database (CARD, version 3.2.8). Detection of an AMR marker is reported if the AMR marker passes a minimum detection threshold and if one or more of the microorganisms associated with the AMR marker is also detected, in alignment with guidance provided by the College of American Pathologists (CAP) MIC.21855. However, reported AMR markers may originate from microorganisms that did not meet detection thresholds or microorganisms not targeted by the test. Association between microorganisms and AMR marker is based on scientific literature and the Comprehensive Antibiotic Research Database Prevalence Data (CARD Prevalence, version 4.0.1) from McMaster University. 3,968 out of 4,371 AMR markers are associated with a microorganism targeted by UPIP. Reported AMR markers have been associated with antimicrobial resistance but may not always indicate phenotypic resistance. Failure to detect AMR markers does not always indicate phenotypic susceptibility. Results should be interpreted in the context of all available information.
AMR - when "Report bacterial AMR markers only when an associated microorganism is reported" is NOT selected
This test detects 4,371 antimicrobial resistance (AMR) markers and reports associations for 72 microorganisms, 185 antimicrobials, and 33 drug classes. AMR markers are based on the Comprehensive Antibiotic Research Database (CARD, version 3.2.8). Association between microorganisms and AMR marker is based on scientific literature and the Comprehensive Antibiotic Research Database Prevalence Data (CARD Prevalence, version 4.0.1) from McMaster University. Detection of an AMR marker is reported if the AMR marker passes a minimum detection threshold, regardless of associated microorganism detection. Reported AMR markers may originate from microorganisms that did not meet detection thresholds or microorganisms not targeted by the test. Reported AMR markers have been associated with antimicrobial resistance but may not always indicate phenotypic resistance. Failure to detect AMR markers does not always indicate phenotypic susceptibility. Results should be interpreted in the context of all available information.
AMR
Linkage between AMR marker, antimicrobial, and drug class is based on the Comprehensive Antibiotic Research Database (CARD, version 3.2.8) from McMaster University, ResFinder (version 2.2.1), NCBI Reference Gene Catalog (version 2023-09-26.1), EUCAST expert rules on indicator agents (2019-2023), and CLSI Performance Standards for Antimicrobial Susceptibility Testing (M100 34th Edition). Not all antimicrobials and drug classes that are listed may be relevant. Detected AMR markers may also confer resistance to antimicrobials and drug classes that are not listed.
AMR
A representative list of associated microorganisms known to harbor the detected or similar AMR markers, based on the Comprehensive Antibiotic Research Database Prevalence Data (CARD Prevalence, version 4.0.1) from McMaster University, can be found in the Associated Microorganisms field.
AMR
Mutations connected with a '+' form an epistatic group. Epistatic groups are two or more mutations that need to be present concurrently to confer the associated resistance.
AMR
All intrinsic resistance described in CLSI Performance Standards for Antimicrobial Susceptibility Testing, M100 34th Edition, Appendix B for detected microorganism(s) is reported. Additional comments regarding CLSI intrinsic resistance definitions may be reported in footnotes specific to the detected microorganism(s). Some intrinsic resistance is described with reference to drug classes rather than specific antimicrobials. Users may reference CLSI Glossary I (Part 1 and Part 2): Class and Subclass Designations and Generic Names for information on how CLSI categorizes antimicrobials and drug classes.
AMR
Confidence of AMR marker detection is shown as High, Medium, or Low and is based on the available sequencing data. High confidence indicates that an AMR marker has 100% protein sequence coverage and 100% protein sequence percent identity (PID). Medium confidence indicates that an AMR marker has ≥90% protein sequence coverage and ≥90% protein sequence percent identity (PID). Low confidence indicates that an AMR marker has ≥60% protein sequence coverage and ≥80% protein sequence percent identity (PID).
Phenotypic group
Targeted microorganisms are classified into three Phenotypic Groups based on general association with urinary tract infections, normal flora, colonization, or contamination from the environment or other sources. Phenotypic grouping DOES NOT INDICATE PATHOGENICITY IN A GIVEN CASE and results need to be interpreted in the context of all available information. Phenotypic Group 1: Microorganisms that are rarely associated with urinary tract infections and may frequently represent normal flora, colonizers, or contaminants. Phenotypic Group 2: Microorganisms that are infrequently associated with urinary tract infections and may frequently represent part of the normal flora, colonizers, or contaminants. Phenotypic Group 3: Microorganisms that are commonly associated with urinary tract infections but may also represent part of the normal flora, colonizers, or contaminants.
Read classification
This test differentiates sequencing reads classified to microorganism and Internal Control regions that are targeted by capture probes (“Targeted Microbial” and “Targeted Internal Control”) from those that are not targeted (“Untargeted”), are low complexity (“Low Complexity”), cannot be unambiguously assigned to one category (“Ambiguous”), or cannot be classified with confidence (“Unclassified”).
Limitations
Non-detected results do not rule out the presence of viruses, bacteria, fungi, parasites, and AMR markers. Contamination with microorganisms is possible during specimen collection, transport, and processing. Closely related microorganisms may be misidentified based on sequence homology to species present in the database. The identification of DNA sequences from a microorganism does not confirm that the identified microorganism is causing symptoms, is viable, or is infectious. Recombinant viral strains may not be reported or may be reported as one or more individual viruses. The Enterobacter cloacae complex may not be reported if targeted species members (Enterobacter cloacae, Enterobacter hormaechei, and Enterobacter cancerogenus) are not present.
Limitations
The best matching allele is reported for each detected AMR gene family. If two or more alleles within the same AMR gene family are detected, only the allele with the higher confidence will be reported as the best match unless multiple alleles have a High confidence interpretation (100% protein sequence coverage and PID). In bacterial strains containing insertion-deletion mutations (indels), there is a risk of false positive or false negative results for other resistance mutations within a region of 100 nucleotides around the indel.
Limitations
Information provided by DRAGEN Microbial Enrichment Plus is based on scientific knowledge and has been curated; however, scientific knowledge evolves and information about associated microorganism and associated resistance may not always be complete and/or correct. Results should be interpreted in the context of all available information. Other sources of data may be required for confirmation.
The Sample Composition bargraphs show the proportion of reads classified to six broad categories for of all samples in the analysis run: Targeted Microbial, Untargeted, Ambiguous, Unclassified, Low Complexity, and Targeted Internal Control (RPIP, UPIP, VSP V2 only).
The Summary Statistics table summarizes sample QC metrics for all samples in the analysis run. Further details on each metric can be found by hovering over each column header.
Individual sample results can be further explored by clicking on "Report" under each sample name in the panel on the left. There are four tabs in the Sample Report: Sample Quality Control, Microorganisms, Antimicrobial Resistance Markers, and User Options.
Version Information is a table with the application version, test type, and test version that were run. Running the latest version of the application is recommended.
Sample Composition is a bargraph showing the proportion of post-quality reads classified to six broad categories for the sample (RPIP, UPIP, VSP V2 only).
Read Classification is a dynamic plot that can be configured to show the following (RPIP, UPIP, VSP V2 only):
Targeted Microbial Reads - Relative (default): Bargraph of post-quality targeted microbial reads belonging to Viral, Bacterial, Fungal, Parasite and AMR categories, relative to post-quality targeted microbial reads only. Percentages are expected to sum to 100%. Hover over an individual bar to display the values.
Targeted Microbial Reads - Absolute: Bargraph of post-quality targeted microbial reads belonging to Viral, Bacterial, Fungal, Parasite and AMR categories for all post-quality reads in the sample overall. Hover over an individual bar to display the values.
Untargeted Reads - Relative: Bargraph of post-quality untargeted reads belonging to untargeted categories, relative to post-quality untargeted reads only. Percentages are expected to sum to 100%. Hover over an individual bar to display the values.
Untargeted Reads - Absolute: Bargraph of post-quality untargeted reads belonging to untargeted categories for all post-quality reads in the sample overall. Hover over an individual bar to display the values.
**Note that accurate sample composition and read classification results rely on selecting the correct enrichment panel. If you run an analysis that is not specific to the enrichment panel (e.g., VSP V2 analysis with VSP-enriched samples), reads from high background viruses that are not targeted by VSP probes (e.g., Measles virus) but that are targeted by VSP V2 probes will be reported as targeted viral reads.
Internal Controls is a table containing supported Internal Control options along with observed RPKM values (RPIP, UPIP, VSP V2 only).
QC Metrics is a table containing sample QC metrics. Dehosting refers to human reads only.
Microorganism results are summarized in tables, separated by type (Viruses, Bacteria, Fungi, Parasites). Each table includes whether the microorganism is predicted present in the sample, as well as various alignment metrics. Further details on each metric can be found by hovering over each column header. The best-match Reference Accession(s) are provided for all RPIP, RVOP/RVEK, VSP, and VSP V2 viruses in the Viruses table. To see all best-match Reference Accession(s), click on the three dots (...) in the table and scroll down the page.
Reference Coverage is a dynamic plot showing the coverage depth across the viral genome for detected RPIP, RVOP/RVEK, VSP, and VSP V2 viruses. Select a virus from the dropdown list to view the coverage plot. Segments are concatenated for segmented viruses, and the targeted regions of the viral genome are indicated for RPIP viruses.
Viral AMR (Variants) is a table with viral AMR variant results for Influenza A/B viruses (RPIP, RVOP/RVEK, VSP, and VSP V2 only)
Bacterial AMR (Genes) is a table witb bacterial AMR gene results (RPIP, UPIP only)
Bacterial AMR (Variants) is a table with bacterial AMR variant results (RPIP, UPIP only)
The User Options table summarizes user options selected during launch of the analysis.
AMR
antimicrobial resistance
mL
milliliter
NAI
neuraminidase inhibitor
NGS
next-generation sequencing
PAI
polymerase acidic endonuclease inhibitor
pangolin
phylogenetic assignment of named global outbreak lineages
RPKM
targeted Reads mapped Per Kilobase of targeted sequence per Million quality-filtered reads
RVEK
Respiratory Virus Enrichment Kit
RVOP
Respiratory Virus Oligo Panel
RUO
For Research Use Only. Not for use in diagnostic procedures.
URL
See https://www.illumina.com/ for additional information.
Quantification
RVOP data analysis using DRAGEN Microbial Enrichment Plus detects 24 viruses and 238 AMR markers based on target enriched next-generation sequencing (NGS) of viral DNA and cDNA sequences. Sequencing data are interpreted by the DRAGEN software platform and viruses that pass detection thresholds are reported. Relative abundance is expressed as proportion of RPKM values.
AMR
This test detects 238 antimicrobial resistance (AMR) markers associated with resistance to Influenza virus neuraminidase inhibitor (NAI) and polymerase acidic protein inhibitor (PAI) in Influenza A virus (H1N1pdm09), Influenza A virus (H1N1), Influenza A virus (H5N1), Influenza A virus (H3N2), Influenza A virus (H3N2; swine-like), Influenza A virus (H7N9), and Influenza B virus. AMR markers and drug associations are based on the World Health Organization (WHO) Influenza virus NAI and PAI Reduced Susceptibility Marker Tables (07 March 2023 version). Detection of an AMR marker is reported if the marker passes a minimum detection threshold and if the Influenza virus associated with the marker is also detected. Reported AMR markers have been associated with antimicrobial resistance but may not always indicate phenotypic resistance. Failure to detect AMR variants does not always indicate phenotypic susceptibility. Results should be interpreted in the context of all available information.
AMR
Mutations connected with a '+' form an epistatic group. Epistatic groups are two or more mutations that need to be present concurrently to confer the associated resistance.
Pango lineage
The most likely Pango (phylogenetic assignment of named global outbreak) lineage is assigned to the majority consensus SARS-CoV-2 genome sequence using pangolin 4.3.1 (Áine O'Toole & Emily Scher et al. 2021 Virus Evolution DOI:10.1093/ve/veab064).
Limitations
Non-detected results do not rule out the presence of viruses and AMR markers. Contamination is possible during specimen collection, transport, and processing. Closely related viruses may be misidentified based on sequence homology to viruses present in the database. The identification of cDNA or DNA sequences from a virus does not confirm that the identified virus is causing symptoms, is viable, or is infectious. Recombinant viral strains may not be reported or may be reported as one or more individual viruses. Should one or more individual viruses be reported for a recombinant viral strain, antiviral resistance results may be inaccurate. In viral strains containing insertion-deletion mutations (indels), there is a risk of false positive or false negative results for other resistance mutations within a region of 100 nucleotides around the indel.
Limitations
Information provided by DRAGEN Microbial Enrichment Plus is based on scientific knowledge and has been curated; however, scientific knowledge evolves and reported information may not always be complete and/or correct. Results should be interpreted in the context of all available information. Other sources of data may be required for confirmation.
Note: Some files may not be generated depending on the selected analysis options and analysis results
Samplename.Panelname.report.json
json
Samplename.Panelname.report.html
html
Samplename.Panelname.viral_variants.vcf
vcf
Viral variant call file describing variant calls between viral consensus genome (or segment) sequences and best-match reference sequences (all RVOP/RVEK, VSP, and VSP V2 viruses, RPIP: SARS-CoV-2 & FluA/B/C only)
Samplename.Panelname.viral_genomes_consensus.fa
fasta
Viral genome (or segment) nucleotide consensus sequence(s) for all viruses reported in the sample (RPIP, RVOP/RVEK, VSP, VSP V2 only)
Samplename.Panelname.viral_targets_consensus.fa
fasta
Viral targeted region nucleotide consensus sequence(s) for all viruses reported in the sample (RPIP only)
Samplename.Panelname.bacterial_amr_nucleotide_consensus.fa
fasta
Bacterial AMR gene nucleotide consensus sequence(s) for all bacterial AMR markers reported in the sample (RPIP, UPIP only)
Samplename.Panelname.bacterial_amr_protein_consensus.fa
fasta
Bacterial AMR gene protein consensus sequence(s) for all bacterial AMR markers reported in the sample (RPIP, UPIP only)
viral_consensus_genomes
Dataset
Directory containing viral genome (or segment) nucleotide consensus sequence(s) per virus reported in the sample (RPIP, RVOP/RVEK, VSP, VSP V2 only)
AnalysisIDnumber.Panelname.results.zip
zip
Compressed file containing all output files for single-click download
AnalysisIDnumber.Panelname.report.xlsx
xlsx
Aggregate Excel report file that summarizes results for all samples across 4 tabs: Samples, Microorganisms, AMR, and Variants. See below for further details
report.html
html
The DRAGEN Microbial Enrichment Plus app outputs a comprehensive sample-level report.json file containing general metadata, version information, sample QC, microorganism, and AMR marker results, as well as detailed test information. The additional convenience file formats generated by the DRAGEN Microbial Enrichment Plus app do not contain novel content.
(*) indicates results generated by the application layer as opposed to the DRAGEN secondary analysis pipeline
Top-Level Node
The top-level section of the report JSON contains general metadata and version information.
.qcReport.sampleQc Node
This section contains information about sample quality control (QC). The fields are relative to .qcReport.sampleQc
.qcReport.enrichmentFactor Node
This section contains information about the enrichment factor calculation and is relevant to RPIP, UPIP, and VSP V2 only. Detection of an appropriate Internal Control is required. The fields are relative to .qcReport.enrichmentFactor
.qcReport.sampleComposition Node
This section contains information about the composition of the sample and is provided for RPIP, UPIP, and VSP V2 only. The fields are relative to .qcReport.sampleComposition
.qcReport.internalControls Node
This section contains information about internal control detection and is relevant to RPIP, UPIP, and VSP V2 only. 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:
.userOptions Node
This section gives information about analysis options specified by the user. The fields are relative to .userOptions
.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[]
.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[]
.targetReport.microorganisms[].variants[] Node
The value of the .targetReport.microorganisms[].variants[] field is an array of objects containing information about viral variants for all RVOP/RVEK, VSP, and 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[]
.targetReport.microorganisms[].pangoLineage[] Node
The value of the .targetReport.microorganisms[].pangoLineage[] field is an array of objects containing information about SARS-CoV-2 Pango lineage prediction results. The following table describes one .targetReport.microorganisms[].pangoLineage[] object. The fields are relative to .targetReport.microorganisms[].pangoLineage[].
.targetReport.microorganisms[].nextclade[] Node
The value of the .targetReport.microorganisms[].nextclade[] field is an array of objects containing information about viral clade assignment results for applicable viruses. The following table describes one .targetReport.microorganisms[].nextclade[] object. The fields are relative to .targetReport.microorganisms[].nextclade[].
.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[]
.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[]
.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[]
.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[]
.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[]
.targetReport.customReferences[].pangoLineage[] Node
The value of the .targetReport.customReferences[].pangoLineage[] field is an array of objects containing information about SARS-CoV-2 Pango lineage prediction results. The following table describes one .targetReport.customReferences[].pangoLineage[] object. The fields are relative to .targetReport.customReferences[].pangoLineage[]
.additionalInformation[] Node
The value of the .additionalInformation[] field is an array of objects containing additional information about the test and data analysis solution. The fields are relative to .additionalInformation[]
A: RPIP, UPIP, RVOP/RVEK, VSP, VSP V2, and Custom infectious disease and microbiology enrichment panels. To analyze the Pan-Coronavirus (Pan-CoV) panel, a custom coronavirus reference sequence database may be specified. The DME+ app is not intended for use with non-infectious disease enrichment panels (such as human exome).
A: The only infectious disease and microbiology enrichment panel without a pre-set DME+ database is the Pan-CoV panel. To analyze Pan-CoV enriched data with the DME+ app, select "Custom Panel" under the "Enrichment Panel" drop-down list and specify a custom coronavirus reference sequence database. Alternatively, we recommend using the DRAGEN Targeted Microbial app.
A: A Basic Basespace Sequence Hub (BSSH) user account is required to access the DME+ app. However, there is no subscription cost for a Basic BSSH account and no compute cost to run the DME+ app. A Basic BSSH account provides 1 TB of free storage. Additional storage may require iCredits.
A: The VSP V2 viral genome sourcing approach aimed at being as inclusive and comprehensive as possible for the 200 targeted human viruses. All viral genomes passing quality filters available as of June 2023 were included in the design, including recombinant and vaccine strains.
A: While there are many possible reasons, one of the most common causes is that the custom database was not formatted correctly. Below are requirements for the custom reference FASTA and (optional) BED file:
Do not exceed the file size limitation: 10 million bases
Do not include duplicate entries
Do not use spaces in the file name; instead use an underscore "_"
File extension must be .fasta or .fa for custom reference FASTA file and .bed for custom reference BED file
If providing a custom reference BED file, the names in the first column of the BED file (chrom) must match the names that appear in the FASTA file (text after > and before the first whitespace character).
A: If enabled, low-quality bases are trimmed from the ends of each read. After trimming, the read is discarded if fewer than 50% of its bases have a quality score greater or equal to q20, the read is shorter than 32 bp, or the read has 5 or more ambiguous bases. It is assumed that appropriate adapter trimming has already been performed.
A: This setting is used as a pre-alignment filtering step for all viral whole-genome sequencing (WGS) panels. The default setting of 5 means that if less than 5 reads classify to the set of reference sequences belonging to a given virus, that virus will not be reported. On the other hand, if 5 or more reads classify to the set of reference sequences belonging to a given virus, read alignment will proceed and alignment-based thresholds will be used to determine whether that virus is reported. The read classification sensitivity can be set as low as 1 or as high as 1000. Lowering the read classification sensitivity threshold below 5 may significantly increase computational run time and is not recommended for most use cases.
A: Pangolin is currently enabled for all enrichment panels except UPIP. For Custom Panel analyses, Pangolin is enabled and will run on custom reference sequences with at least 3% coverage that meet these naming conventions:
If only a FASTA file is provided, Pangolin will run on sequences that have a header containing either SARS-CoV-2 or NC_045512
If both a FASTA and BED file are provided, Pangolin will run on sequences where the first column (chrom) contains NC_045512 or the fourth column (genomeName) contains SARS-CoV-2
A: When enabled, a Nextclade analysis using the specified dataset(s) is run for the following microorganisms, as applicable:
A: The RPIP, UPIP, and VSP V2 enrichment panels contain probes targeting commercially available Internal Controls. See the table below for Internal Control options compatible with RPIP, UPIP, and VSP V2. It is recommended to spike each sample prior to extraction with Enterobacteria phage T7 at 1.21 x 10^7 copies/mL of sample.
*Quantitative Internal Control concentration must be provided
A: See the table below. Consensus sequence bases without aligned read support are indicated by "N" bases.
A: To evaluate microorganism absolute quantification results, it is recommended to perform experiments using the relevant sample type and full sequencing workflow (including extraction) and to compare results obtained from the DME+ app with those from digital droplet PCR (ddPCR) and/or quantitative PCR (qPCR) assays. A per-microorganism absolute quantification correction factor can be applied to DME+ results as needed.
A: Yes. Not all antimicrobials and drug classes that are listed may be relevant. Detected AMR markers may also confer resistance to antimicrobials and drug classes that are not listed. Linkage between bacterial AMR marker, antimicrobial, and drug class is based on the Comprehensive Antibiotic Research Database (CARD, version 3.2.8) from McMaster University, ResFinder (version 2.2.1), NCBI Reference Gene Catalog (version 2023-09-26.1), EUCAST expert rules on indicator agents (2019-2023), and CLSI Performance Standards for Antimicrobial Susceptibility Testing (M100 34th Edition). Linkage between viral AMR marker, antimicrobial, and drug class is based on the publications provided in the JSON report - see the PubMed IDs (pmids) field.
A: Extended-spectrum beta-lactamase (ESBL) and Carbapenemase flags are assigned based on the antimicrobials and drug classes associated with each bacterial AMR marker. An ESBL flag is reported if a 3rd, 4th, or 5th generation cepholosporin OR a beta-lactam + beta-lactamase inhibitor combination is contained in the list of associated antimicrobials or drug classes. A carbapenemase flag is reported if a carbapenem is contained in the list of associated antimicrobials or drug classes. The logic for each of these flags is decoupled, such that a marker can be reported with both flags if the associated antimicrobial or drug class metadata indicates both ESBL and carbapenemase activity.
A: For complex samples or samples with the majority of nucleic acid being host/untargeted, while 100-1000X more targeted reads and sensitivity over a shotgun/pre-enriched library is expected, typically targeted reads will still only represent a minority of the overall sequencing reads. Notably, RPIP, UPIP, and VSP V2 support various Internal Control options that can be spiked into samples prior to extraction to enable automated calculation of an enrichment factor sample QC metric.
A: The % Targeted Microbial Reads is calculated using a kmer-based classification approach that is intended to give a quick, high-level overview of sample composition. The Aligned Read Count values for microorganisms are calculated in a separate pipeline step using microorganism-specific reference sequence alignment as opposed to broad, categorical, kmer-based classification. Reads that were unclassified or that were classified as low-complexity or ambiguous may actually align to reference sequences. It is also possible for a read to align to a reference sequence of more than one microorganism, for example in a conserved region.
A: FASTQ files previously run through other apps can be re-analyzed using the DME+ app. Results from other apps may not be identical to results from the DME+ app, most notably because of the expanded databases used in DME+.
Launch the DRAGEN Microbial Enrichment Plus BaseSpace app, which can be found in the "Dragen" and "Infectious Disease + Microbiology" app collections.
Enter a name for the Analysis.
Choose either “Biosample” or “Project” as input type. When a Project is selected, the app will attempt to find all FASTQ files in that Project and run analyses on them. There is no FASTQ file limitation when reading Biosamples from a Project. However, 99 associated FASTQ files is the maximum allowed per analysis when providing Biosample input from a list.
Under "Enrichment Panel Microorganism Reporting List", select from the available list to report All microorganisms (default), specify a Pre-defined subset of microorganisms (RPIP, UPIP only), or specify a User-defined microorganism reporting list and reporting thresholds.
If Pre-defined is selected, the Pre-defined specification section is enabled to allow specification of a pre-defined subset of microorganisms for the selected Enrichment Panel. This option is only available for RPIP and UPIP.
Analysis Options:
Perform read QC (Quality Control)
If checked, reads are pre-processed using quality metrics before analysis.
If unchecked, read quality metrics are calculated, but reads are not trimmed or filtered before analysis.
Report bacterial AMR markers only
If checked, only bacterial AMR markers but no microorganisms are reported
This option is disabled if RVOP/RVEK, VSP, VSP V2 or Custom Panel is selected
This option is disabled if the "Report bacterial AMR markers only when an associated microorganism is reported" option is enabled
Report bacterial AMR markers only when an associated microorganism is reported
If checked, detected bacterial AMR markers are reported if the bacterial AMR marker passes a minimum reporting threshold and one or more associated microorganisms are also detected and reported
If unchecked, detected bacterial AMR markers are reported if the bacterial AMR marker passes a minimum reporting threshold
This option is disabled if RVOP/RVEK, VSP, VSP V2 or Custom Panel is selected
Report microorganisms and/or AMR markers that are below threshold
If checked, microorganisms and/or AMR markers below reporting thresholds are included in reports
If unchecked, only microorganisms and/or AMR markers above reporting thresholds are included in reports
This option is disabled if Custom Panel is selected
This option is disabled if the "Report bacterial AMR markers only when an associated microorganism is reported" option is enabled
Specify "Read classification sensitivity". This setting is used as a pre-alignment filtering step for RVOP/RVEK, VSP, and VSP V2 only. The default setting of 5 means that if less than 5 reads classify to the set of reference sequences belonging to a given virus, that virus will not be reported. On the other hand, if 5 or more reads classify to the set of reference sequences belonging to a given virus, read alignment will proceed and alignment-based thresholds will be used to determine whether that virus is reported. The read classification sensitivity can be set as low as 1 or as high as 1000. Lowering the read classification sensitivity threshold below 5 may significantly increase computational run time and is not recommended for most use cases.
Pangolin is currently enabled for all enrichment panels besides UPIP. For Custom Panel analyses, Pangolin will run on custom reference sequences with at least 3% coverage that meet these naming conventions:
If only a FASTA file is provided, Pangolin will run on sequences that have a header containing either SARS-CoV-2 or NC_045512
If both a FASTA and BED file are provided, Pangolin will run on sequences where the first column (chrom) contains NC_045512 or the fourth column (genomeName) contains SARS-CoV-2
Optionally, enable Nextclade to run when one of the following microorganisms is detected (RPIP, RVOP/RVEK, VSP, VSP V2 only):
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
Influenza A virus (H1N1)
Influenza A virus (H3N2)
Influenza A virus (H5N1)
Influenza A virus (H5N6)
Influenza A virus (H5N8)
Influenza B virus (B/Victoria/2/87-like)
Influenza B virus (B/Yamagata/16/88-like)
Human immunodeficiency virus 1 (HIV-1)
Human respiratory syncytial virus A (HRSV-A)
Human respiratory syncytial virus B (HRSV-B)
Monkeypox virus (MPV)
Measles virus (MV)
Dengue virus (DENV), Dengue virus type 1 (DENV-1), Dengue virus type 2 (DENV-2), Dengue virus type 3 (DENV-3), Dengue virus type 4 (DENV-4)
Select a quantitative Internal Control (IC) from the available list (RPIP, UPIP, VSP V2 only). If the quantitative IC is set to NONE, which is the default, the IC concentration value is ignored. For VSP V2, the only valid quantitative IC selections are:
NONE
Enterobacteria phage T7
Escherichia virus T4
Escherichia Virus MS2
Armored RNA Quant Internal Process Control
Enter Internal Control (IC) concentration as an integer in the following scientific notation format: "#.## x 10^#". **An incorrect quantitative IC or incorrect IC concentration will result in inaccurate microorganism absolute quantification results.
Select the Project where the Analysis Output should be saved.
Comprehensive report file. See for further details
Visual report file. See for further details
Visual report file. See for further details
A: Upload these files to a BSSH project before launching the DME+ app. It will then be possible to select these files in the "Select Dataset File(s)" browser in the app. Please see and reach out to techsupport@illumina.com with any unresolved upload issues.
A: See the "Virus Types Captured" column of the "Microorganisms" table in the .
A: The full viral genome is targeted for all RVOP/RVEK, VSP, and VSP V2 viruses. For RPIP viruses, see the "Percent Genome Targeted" column of the "Microorganisms" table in the . No more than ~1% of bacterial, fungal, and parasitic genomes are targeted by RPIP or UPIP.
See for further details.
A: Ensure that the correct microorganism reporting file was uploaded and used. We recommend saving the updated microorganism reporting file with a new name. Rows with microorganism names that are not of interest can be deleted, but do not add any new columns or delete any columns from the provided template. Similarly, do not change or remove any text from the header row. Also, please note that the "kmer_read_count" metric is only valid with the UPIP panel. See for further details.
A: Not necessarily. The microbe of interest may be present in the sample, but the DME+ app may not have reported it because the detection metrics fell below the default reporting thresholds. If it is suspected that this may be the case, select the "Report microorganisms and/or AMR markers that are below threshold" option. A user-defined microorganism reporting file can also be specified on a microorganism-by-microorganism basis using multiple parameters should more sensitive reporting be required for a given use case. See for further details.
A: Multiple parameters are used to determine whether the sequencing data for a given microorganism is sufficient for a positive call. These may include the horizontal coverage, median read depth, normalized read count, average nucleotide identity, etc of the microorganism and/or other genetically related microorganisms. The default reporting thresholds are different for different microorganisms, as microorganisms with close genetic neighbors generally require more stringent reporting thresholds than genetically distinct microorganisms. As with most tests and prediction algorithms, the default reporting thresholds are intended to balance the trade-off between analytical sensitivity and specificity. Should a given use case require more sensitive or specific reporting, a user-defined microorganism reporting file can be specified on a microorganism-by-microorganism basis using multiple parameters. See for further details. Additionally, the "Report microorganisms and/or AMR markers that are below threshold" option can be enabled.
A: Mathematically, any result with a horizontal coverage of <50% will have a median depth of 0 (50% or more of the nucleotide positions have a depth of 0). Low coverage results could represent true low positives (the most likely reason) or non-specific results, contamination, etc. If maximum confidence is required for a given use case, stricter microorganism reporting thresholds can be specified on a microorganism-by-microorganism basis using multiple parameters. See for further details.
A: See the "Has Tiered Reporting" and "Reporting Tier" columns of the "Microorganisms" table in the for RPIP, RVOP/RVEK, VSP, and VSP V2 to select and see which viruses are reported as part of a tiered reporting group. Membership in a tiered reporting group means that a hierarchical relationship is pre-built into the database and the most granular tier level passing reporting thresholds is reported. For example, if Influenza B virus (B/Victoria/2/87-like) or Influenza B virus (B/Yamagata/16/88-like) are reported in a sample then the less granular Influenza B virus reporting name will NOT be reported. Tiered reporting group membership is especially relevant when specifying a user-defined microorganism reporting file as including the entire tiered reporting group is necessary to preserve tiered reporting logic.
A: See the "Has Tiered Reporting" and "Lineage/Clade Prediction" columns of the "Microorganisms" table in the for RPIP, RVOP/RVEK, VSP, and VSP V2. Consensus sequence and best match reference accession are also provided for RPIP, RVOP/RVEK, VSP, and VSP V2 viruses. Subtype information may be possible to infer from the consensus sequence (e.g. by Blast) or from the best match reference accession (if annotated in NCBI). Consensus sequence can also be used as input to downstream viral typing tools.
A: Viral genomes are orders of magnitude smaller and thus computationally much "cheaper" to align to than bacterial, fungal, and parasitic genomes. In the case of RVOP/RVEK, VSP, and VSP V2, the full viral genome is targeted for all viruses. For RPIP viruses, see the "Percent Genome Targeted" column of the "Microorganisms" table in the . While not visualized in the HTML report at this time, the DME+ does contain coverage depth vector information for all microorganism targeted regions (viruses, bacteria, fungi, and parasites). See: .targetReport.microorganisms[].condensedDepthVector[], which is the read depth across the targeted microorganism reference sequences, condensed (if needed) into 256 bins.
Select a target-capture Enrichment Panel for the appropriate analysis options and default settings to populate. Only one enrichment panel can be selected per analysis. If Custom Panel is selected, the "Custom panel specification" section is enabled to allow entry of a reference FASTA file and (optionally) a reference BED file. See for further details.
If User-defined is selected, the User-defined specification section is enabled to allow entry of a microorganism reporting list and reporting thresholds file in TSV or XLSX format. See for further details.
.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 set to “POS” if the substring “PosCon” is found in the sample name, “NEG” if the substring “NegCon” is found, or “BLANK” if the substring “controlBlk” is found. Otherwise, it is set to “-”
.dragenVersion
DRAGEN release version
.analysisPipelineVersion
Analysis Pipeline release version
.testType
Type of test panel ("RPIP", "UPIP", "RVOP", "VSPv1", "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
.results*
High level result: “One or more potential pathogens predicted” or ”No potential pathogens predicted”
.appVersion*
DRAGEN Microbial Enrichment plus application release version
.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
.value
Enrichment factor value reflecting how well targeted regions were enriched
.category
Enrichment factor category: "poor", "fair", "good", or "not calculated"
.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
.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
(RVOP/RVEK, VSP, 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
.belowThresholdEnabled*
Boolean indicating if microorganisms and/or AMR markers below detection thresholds are reported
.bacterialAmrMarkersOnly*
(RPIP, UPIP only) Boolean indicating if only bacterial AMR markers are reported
.bacterialAmrMarkerMicroorganismRequired*
(RPIP, UPIP only) Boolean indicating if bacterial AMR markers are reported only when an associated microorganism is reported
.preDefinedMicroorganismReportingList*
(RPIP, UPIP only) Pre-defined microorganism reporting list, if specified
.userDefinedMicroorganismReportingListUsed*
Boolean indicating if a user-defined microorganism reporting file is specified
.userDefinedMicroorganismReportingListFile*
Name of the user-defined microorganism reporting file, if specified
.providedAnalysisName*
User-provided analysis name
.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, RVOP/RVEK, VSP, 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
.consensusTargetSequences.scaledDepthVector*
Read depth across the targeted region reference sequence, condensed and scaled such that the longest targeted region for the microorganism has a maximum length of 256 bins
.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
.predictionInformation.userDefined*
User-defined reporting prediction logic for microorganism, if specified
.variants
(all RVOP/RVEK, VSP, and VSP V2 viruses, RPIP: SARS-CoV-2 & FluA/B/C only) Information about viral variants. See below for details
.comments*
List of additional information regarding the microorganism
.abundance*
Relative abundance of the microorganism within the microorganism class
.pangoLineage*
(SARS-CoV-2 only) Information about SARS-CoV-2 Pango lineage prediction results. See below for details
.nextclade*
(applicable viruses only) Information about viral clade assignment results. See below for details
.potentialAmrDetected*
(Bacteria only) Potential AMR detection flag for microorganism. Can be "Yes", “Not Detected”, or “n/a”
.potentialAmrPredicted*
(Bacteria only) Potential AMR prediction flag for microorganism. Can be "Yes", “Not Predicted”, or “n/a”
.flags*
(Bacteria only) Flag for potential resistance to an important drug class ("Potential ESBL", "Potential Carbapenemase")
.intrinsicResistance*
(Bacteria only) List of antimicrobials to which the reported bacteria is intrinsically resistant, based on CLSI Performance Standards for Antimicrobial Susceptibility Testing, M100 34th Edition, Appendix B
.intrinsicResistanceDrugClasses*
(Bacteria only) List of drug classes to which the reported bacteria is intrinsically resistant, based on CLSI Performance Standards for Antimicrobial Susceptibility Testing, M100 34th Edition, Appendix B
.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
.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
.category*
Variant category ("Viral Variant; Known AMR", "Viral Variant")
.comments*
List of additional information regarding the variant
.gene*
(SARS-CoV-2, Flu A/B/C only) Gene name
.product*
Protein product of gene
.annotation*
Type of change (e.g., "Nonsynonymous Variant")
.aachange*
Amino acid change associated with variant
.epistaticGroups*
List of epistatic groups variant is associated with
.standardNomenclatureEpistaticGroups*
(Flu A/B only) List of epistatic groups variant is associated with using standard nomenclature coordinates
.standardNomenclatureAaChange*
(Flu A/B only) Amino acid change associated with variant using standard nomenclature coordinates
.standardNomenclatureAccession*
(Flu A/B only) NCBI accession of the reference sequence used to establish standard nomenclature coordinates
.drugClasses*
List of drug classes variant is predicted to confer resistance to
.representativeAntimicrobials*
List of representative antimicrobials variant is predicted to confer resistance to
.inhibitionLevel*
(Flu A/B only) Level of inhibition per cited publications (see pmids)
.pmids*
PubMed IDs of publications associated with variant
Field
Description [Source]
.lineage*
From Pangolin: "The most likely lineage assigned to a given sequence based on the inference engine used and the SARS-CoV-2 diversity designated. This assignment may be sensitive to missing data at key sites"
.conflict*
From Pangolin: "In the pangoLEARN model, a given sequence gets assigned to the most likely category based on known diversity. If a sequence can fit into more than one category, the conflict score will be greater than 0 and reflect the number of categories the sequence could fit into. If the conflict score is 0, this means that within the current decision tree there is only one category that the sequence could be assigned to"
.ambiguityScore*
From Pangolin: "This score is a function of the quantity of missing data in a sequence. It represents the proportion of relevant sites in a sequnece which were imputed to the reference values. A score of 1 indicates that no sites were imputed, while a score of 0 indicates that more sites were imputed than were not imputed. This score only includes sites which are used by the decision tree to classify a sequence"
.version*
Version of the PUSHER database
.pangolinVersion*
Version of the Pangolin software
Field
Description [Source]
.sequenceName*
Name of the sequence
.referenceAccession*
Reference accession
.overallStatus*
Overall quality control status
.clade*
Assigned clade
.pangoLineage*
Pango lineage assigned by Nextclade
.cladeWho*
World Health Organization (WHO) nomenclature
.substitutions*
Total number of detected nucleotide substitutions
.totalNonACGTNs*
Total number of detected ambiguous nucleotides (nucleotide characters that are not A, C, G, T, N)
.totalMissing*
Total number of detected missing nucleotides (nucleotide character N)
.coverage*
Proportion of consensus genome (or segment) sequence bases that aligned to reference accession
.totalInsertions*
Total number of inserted nucleotide bases
.totalFrameShifts*
Total number of detected frame shifts
.stopCodons*
Total number of detected stop codons
.version*
Version of the Nextclade software
.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
.flags*
Flag indicating AMR marker is an extended-spectrum beta-lactamase (ESBL) or carbapenemase (Carbapenemase)
.representativeAntimicrobials*
List of representative antimicrobials the AMR marker is predicted to confer resistance to
.drugClasses*
List of drug classes the AMR marker is predicted to confer resistance to
.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
.representativeAntimicrobials*
List of representative antimicrobials variant is predicted to confer resistance to
.drugClasses*
List of drug classes variant is predicted to confer resistance to
.confidenceLevel*
(MTB only) Confidence level is given for Mycobacterium tuberculosis variants if provided by the WHO Catalogue of mutations in Mycobacterium tuberculosis (Final Grading Confidence; for rpoB only), or the Comprehensive Antibiotic Resistance Database (CARD), as part of a confidence model for AMR developed by the Relational Sequencing Tuberculosis Data Platform (ReSeqTB)
.pmids*
PubMed IDs of publications associated with variant
.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
.pangoLineage*
Array of objects with information about SARS-CoV-2 Pango lineage prediction results. See below for details
.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
.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
Field
Description [Source]
.lineage*
The most likely lineage assigned to a given sequence based on the inference engine used and the SARS-CoV-2 diversity designated. This assignment may be is sensitive to missing data at key sites
.conflict*
In the pangoLEARN model, a given sequence gets assigned to the most likely category based on known diversity. If a sequence can fit into more than one category, the conflict score will be greater than 0 and reflect the number of categories the sequence could fit into. If the conflict score is 0, this means that within the current decision tree there is only one category that the sequence could be assigned to
.ambiguityScore*
This score is a function of the quantity of missing data in a sequence. It represents the proportion of relevant sites in a sequnece which were imputed to the reference values. A score of 1 indicates that no sites were imputed, while a score of 0 indicates that more sites were imputed than were not imputed. This score only includes sites which are used by the decision tree to classify a sequence
.version*
Version of the PUSHER database
.pangolinVersion*
Version of the Pangolin software
.abbreviations*
Information about abbreviations relevant to test
.abbreviations.abbreviation*
Abbreviation
.abbreviations.definition*
Abbreviation definition
.interpretiveData*
Information about test
.interpretiveData.header*
Test information category
.interpretiveData.paragraph*
Test information text
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
SARS-CoV-2 (relative to Wuhan-Hu-1/2019)
Official
Influenza A virus (H1N1)
Influenza A H1N1pdm HA (relative to A/Wisconsin/588/2019) & Influenza A H1N1pdm NA (relative to A/Wisconsin/588/2019)
Official
Influenza A virus (H3N2)
Influenza A H3N2 HA (relative to A/Darwin/6/2021) & Influenza A H3N2 NA (relative to A/Darwin/6/2021)
Official
Influenza B virus (B/Victoria/2/87-like)
Influenza B Victoria HA (relative to B/Brisbane/60/2008)
Official
Influenza B virus (B/Yamagata/16/88-like)
Influenza B Yamagata HA (relative to B/Wisconsin/01/2010)
Official
Human respiratory syncytial virus A (HRSV-A)
RSV-A
Official
Human respiratory syncytial virus B (HRSV-B)
RSV-B
Official
Monkeypox virus (MPV)
Mpox virus (All Clades)
Official
Measles virus (MV)
Measles virus N450 (WHO-2012)
Official
Dengue virus (DENV), Dengue virus type 1 (DENV-1), Dengue virus type 2 (DENV-2), Dengue virus type 3 (DENV-3), Dengue virus type 4 (DENV-4)
Dengue virus (All Serotypes)
Official
Human immunodeficiency virus 1 (HIV-1)
HIV-1 (relative to HXB2)
Community
Influenza A virus (H5N1)
Influenza A H5Nx HA (relative to A/Goose/Guangdong/1/96)
Community
Influenza A virus (H5N6)
Influenza A H5Nx HA (relative to A/Goose/Guangdong/1/96)
Community
Influenza A virus (H5N8)
Influenza A H5Nx HA (relative to A/Goose/Guangdong/1/96)
Community
Allobacillus halotolerans
X
X
X
X
X
Armored RNA Quant Internal Process Control
X
X
X
X
X
Enterobacteria phage T7
X
X
X
X
X
X
Recommended IC concentration = 1.21 x 10^7 copies/mL of sample
Escherichia virus MS2
X
X
X
X
X
X
Escherichia virus Qbeta
X
X
X
X
X
Escherichia virus T4
X
X
X
X
X
X
Imtechella halotolerans
X
X
X
X
X
Phocid alphaherpesvirus
X
X
X
X
X
Phocine morbillivirus
X
X
X
X
X
Truepera radiovictrix
X
X
X
X
X
Read de-duplication
Not performed
Depth threshold for consensus sequence generation
1x
Depth threshold for variant calling
5x
Minimum minor allele frequency
20%
AMR
antimicrobial resistance
CLSI
Clinical and Laboratory Standards Institute
ESBL
extended spectrum beta-lactamase
EUCAST
European Committee on Antimicrobial Susceptibility Testing
mL
milliliter
NAI
neuraminidase inhibitor
NGS
next-generation sequencing
PAI
polymerase acidic endonuclease inhibitor
pangolin
phylogenetic assignment of named global outbreak lineages
RPIP
Respiratory Pathogen ID/AMR Panel
RPKM
targeted Reads mapped Per Kilobase of targeted sequence per Million quality-filtered reads
RUO
For Research Use Only. Not for use in diagnostic procedures.
URL
See https://www.illumina.com/ for additional information.
Quantification - when a quantitative Internal Control {ic_name} and concentration {ic_concentration} is specified
RPIP data analysis using DRAGEN Microbial Enrichment Plus detects 41 viruses, 187 bacteria, 53 fungi, and 4,079 AMR markers, unless filtered reporting options are selected, based on target enriched next-generation sequencing (NGS) of microorganism DNA and cDNA sequences. Sequencing data are interpreted by the DRAGEN software platform and microorganisms that pass detection thresholds are reported. Absolute quantification assumes use of {ic_name} as an Internal Control spiked at {ic_concentration} copies/mL of sample. Relative abundance is calculated based on absolute quantities and is expressed as proportion of absolute quantities within each pathogen class (i.e., bacteria, viruses, fungi). If RPKM for the Internal Control is zero, no absolute quantification is provided, and relative abundance is expressed as proportion of microorganism RPKM values within each pathogen class.
Quantification - when a quantitative Internal Control is NOT specified
RPIP data analysis using DRAGEN Microbial Enrichment Plus detects 41 viruses, 187 bacteria, 53 fungi, and 4,079 AMR markers, unless filtered reporting options are selected, based on target enriched next-generation sequencing (NGS) of microorganism DNA and cDNA sequences. Sequencing data are interpreted by the DRAGEN software platform and microorganisms that pass detection thresholds are reported. Relative abundance is expressed as proportion of microorganism RPKM values within each pathogen class (i.e., bacteria, viruses, fungi). Internal Control not specified; no absolute quantification provided.
AMR - when "Report bacterial AMR markers only when an associated microorganism is reported" is selected
This test detects 4,079 antimicrobial resistance (AMR) markers and reports associations for 99 microorganisms, 181 antimicrobials, and 35 drug classes, unless filtered reporting options are selected. Bacterial AMR markers are based on the Comprehensive Antibiotic Research Database (CARD, version 3.2.8) and viral AMR markers are based on World Health Organization (WHO) Influenza virus neuraminidase inhibitor (NAI) and polymerase acidic protein inhibitor (PAI) Reduced Susceptibility Marker Tables (07 March 2023 version). Detection of an AMR marker is reported if the AMR marker passes a minimum detection threshold and if one or more of the microorganisms associated with the AMR marker is also detected, in alignment with guidance provided by the College of American Pathologists (CAP) MIC.21855. However, reported AMR markers may originate from microorganisms that did not meet detection thresholds or microorganisms not targeted by the test. Association between microorganisms and bacterial AMR marker is based on scientific literature and the Comprehensive Antibiotic Research Database Prevalence Data (CARD Prevalence, version 4.0.1) from McMaster University. Reported AMR markers have been associated with antimicrobial resistance but may not always indicate phenotypic resistance. Failure to detect AMR markers does not always indicate phenotypic susceptibility. Results should be interpreted in the context of all available information.
AMR - when "Report bacterial AMR markers only when an associated microorganism is reported" is NOT selected
This test detects 4,079 antimicrobial resistance (AMR) markers and reports associations for 99 microorganisms, 181 antimicrobials, and 35 drug classes. Bacterial AMR markers are based on the Comprehensive Antibiotic Research Database (CARD, version 3.2.8) and viral AMR markers are based on World Health Organization (WHO) Influenza virus neuraminidase inhibitor (NAI) and polymerase acidic protein inhibitor (PAI) Reduced Susceptibility Marker Tables (07 March 2023 version). Association between microorganisms and bacterial AMR marker is based on scientific literature and the Comprehensive Antibiotic Research Database Prevalence Data (CARD Prevalence, version 4.0.1) from McMaster University. Detection of a bacterial AMR marker is reported if the marker passes a minimum detection threshold, regardless of associated microorganism detection. Reported AMR markers may originate from microorganisms that did not meet detection thresholds or microorganisms not targeted by the test. Reported AMR markers have been associated with antimicrobial resistance but may not always indicate phenotypic resistance. Failure to detect AMR markers does not always indicate phenotypic susceptibility. Results should be interpreted in the context of all available information.
AMR
Linkage between bacterial AMR marker, antimicrobial, and drug class is based on the Comprehensive Antibiotic Research Database (CARD, version 3.2.8) from McMaster University, ResFinder (version 2.2.1), NCBI Reference Gene Catalog (version 2023-09-26.1), EUCAST expert rules on indicator agents (2019-2023), and CLSI Performance Standards for Antimicrobial Susceptibility Testing (M100 34th Edition). Linkage between viral AMR marker, antimicrobial, and drug class is based on the publications provided in the JSON report - see PubMed IDs (pmids) field. Not all antimicrobials and drug classes that are listed may be relevant. Detected AMR markers may also confer resistance to antimicrobials and drug classes that are not listed.
AMR
A representative list of associated microorganisms known to harbor the detected or similar bacterial AMR markers, based on the Comprehensive Antibiotic Research Database Prevalence Data (CARD Prevalence, version 4.0.1) from McMaster University, can be found in the Associated Microorganisms field.
AMR
Mutations connected with a '+' form an epistatic group. Epistatic groups are two or more mutations that need to be present concurrently to confer the associated resistance.
AMR
All intrinsic resistance described in CLSI Performance Standards for Antimicrobial Susceptibility Testing, M100 34th Edition, Appendix B for detected microorganism(s) is reported. Additional comments regarding CLSI intrinsic resistance definitions may be reported in footnotes specific to the detected microorganism(s). Some intrinsic resistance is described with reference to drug classes rather than specific antimicrobials. Users may reference CLSI Glossary I (Part 1 and Part 2): Class and Subclass Designations and Generic Names for information on how CLSI categorizes antimicrobials and drug classes.
AMR
Confidence of bacterial AMR marker detection is shown as High, Medium, or Low and is based on the available sequencing data. High confidence indicates that a bacterial AMR marker has 100% protein sequence coverage and 100% protein sequence percent identity (PID). Medium confidence indicates that a bacterial AMR marker has ≥90% protein sequence coverage and ≥90% protein sequence percent identity (PID). Low confidence indicates that a bacterial AMR marker has ≥60% protein sequence coverage and ≥80% protein sequence percent identity (PID).
Phenotypic group
Targeted microorganisms are classified into three Phenotypic Groups based on general association with normal flora, colonization, or contamination from the environment or other sources, as well as based on general association with disease. Phenotypic grouping DOES NOT INDICATE PATHOGENICITY IN A GIVEN CASE and results need to be interpreted in the context of all available information. Phenotypic Group 1: Microorganisms that are frequently considered part of the normal flora, colonizers, or contaminants but may be associated with disease in certain settings. Phenotypic Group 2: Microorganisms that may represent normal flora, colonizers, or contaminants but that are frequently associated with disease. Phenotypic Group 3: Microorganisms that are not generally considered part of the normal flora, colonizers, or contaminants and are generally considered to be associated with disease.
Pango lineage
The most likely Pango (phylogenetic assignment of named global outbreak) lineage is assigned to the majority consensus SARS-CoV-2 genome sequence using pangolin 4.3.1 (Áine O'Toole & Emily Scher et al. 2021 Virus Evolution DOI:10.1093/ve/veab064).
Read classification
This test differentiates sequencing reads classified to microorganism and Internal Control regions that are targeted by capture probes (“Targeted Microbial” and “Targeted Internal Control”) from those that are not targeted (“Untargeted”), are low complexity (“Low Complexity”), cannot be unambiguously assigned to one category (“Ambiguous”), or cannot be classified with confidence (“Unclassified”).
Limitations
Non-detected results do not rule out the presence of viruses, bacteria, fungi, and AMR markers. Contamination with microorganisms is possible during specimen collection, transport, and processing. Closely related microorganisms may be misidentified based on sequence homology to species present in the database. The identification of cDNA or DNA sequences from a microorganism does not confirm that the identified microorganism is causing symptoms, is viable, or is infectious. Recombinant viral strains may not be reported or may be reported as one or more individual viruses. Should one or more individual viruses be reported for a recombinant viral strain, antiviral resistance results may be inaccurate.
Limitations
The best matching allele is reported for each detected bacterial AMR gene family. If two or more alleles within the same bacterial AMR gene family are detected, only the allele with the higher confidence will be reported as the best match unless multiple alleles have a High confidence interpretation (100% coverage and PID). In strains containing insertion-deletion mutations (indels), there is a risk of false positive or false negative results for other resistance mutations within a region of 100 nucleotides around the indel.
Limitations
Information provided by DRAGEN Microbial Enrichment Plus is based on scientific knowledge and has been curated; however, scientific knowledge evolves and information about associated microorganism and associated resistance may not always be complete and/or correct. Results should be interpreted in the context of all available information. Other sources of data may be required for confirmation.
NGS
next-generation sequencing
pangolin
phylogenetic assignment of named global outbreak lineages
RUO
For Research Use Only. Not for use in diagnostic procedures.
URL
See https://www.illumina.com/ for additional information.
Pango lineage
The most likely Pango (phylogenetic assignment of named global outbreak) lineage is assigned to the majority consensus SARS-CoV-2 genome sequence using pangolin 4.3.1 (Áine O'Toole & Emily Scher et al. 2021 Virus Evolution DOI:10.1093/ve/veab064).
Limitations
Custom panel data analysis using DRAGEN Microbial Enrichment Plus aligns human-dehosted next-generation sequencing (NGS) reads to reference sequences. Contamination with microorganisms is possible during specimen collection, transport, and processing. Reads from closely related microorganisms may align to reference sequences based on sequence homology. Alignment of reads to a microorganism does not confirm that the microorganism is causing symptoms, is viable, or is infectious. Results should be interpreted in the context of all available information. Other sources of data may be required for confirmation.
AMR
antimicrobial resistance
mL
milliliter
NAI
neuraminidase inhibitor
NGS
next-generation sequencing
PAI
polymerase acidic endonuclease inhibitor
pangolin
phylogenetic assignment of named global outbreak lineages
RPKM
targeted Reads mapped Per Kilobase of targeted sequence per Million quality-filtered reads
VSP
Viral Surveillance Panel
RUO
For Research Use Only. Not for use in diagnostic procedures.
URL
See https://www.illumina.com/ for additional information.
Quantification - when a quantitative Internal Control {ic_name} and concentration {ic_concentration} is specified
VSP (generation 2) data analysis using DRAGEN Microbial Enrichment Plus detects 200 viruses and 238 AMR markers based on target enriched next-generation sequencing (NGS) of viral DNA and cDNA sequences. Sequencing data are interpreted by the DRAGEN software platform and viruses that pass detection thresholds are reported. Absolute quantification assumes use of {ic_name} as an Internal Control spiked at {ic_concentration} copies/mL of sample. Relative abundance is calculated based on absolute quantities and is expressed as proportion of absolute quantities. If RPKM for the Internal Control is zero, no absolute quantification is provided, and relative abundance is expressed as proportion of RPKM values.
Quantification - when a quantitative Internal Control is NOT specified
VSP (generation 2) data analysis using DRAGEN Microbial Enrichment Plus detects 200 viruses and 238 AMR markers based on target enriched next-generation sequencing (NGS) of viral DNA and cDNA sequences. Sequencing data are interpreted by the DRAGEN software platform and viruses that pass detection thresholds are reported. Relative abundance is expressed as proportion of RPKM values. Internal Control not specified; no absolute quantification provided.
AMR
This test detects 238 antimicrobial resistance (AMR) markers associated with resistance to Influenza virus neuraminidase inhibitor (NAI) and polymerase acidic protein inhibitor (PAI) in Influenza A virus (H1N1pdm09), Influenza A virus (H1N1), Influenza A virus (H5N1), Influenza A virus (H3N2), Influenza A virus (H3N2; swine-like), Influenza A virus (H7N9), and Influenza B virus. AMR markers and drug associations are based on the World Health Organization (WHO) Influenza virus NAI and PAI Reduced Susceptibility Marker Tables (07 March 2023 version). Detection of an AMR marker is reported if the marker passes a minimum detection threshold and if the Influenza virus associated with the marker is also detected. Reported AMR markers have been associated with antimicrobial resistance but may not always indicate phenotypic resistance. Failure to detect AMR variants does not always indicate phenotypic susceptibility. Results should be interpreted in the context of all available information.
AMR
Mutations connected with a '+' form an epistatic group. Epistatic groups are two or more mutations that need to be present concurrently to confer the associated resistance.
Read classification
This test differentiates sequencing reads classified to microorganism and Internal Control regions that are targeted by capture probes (“Targeted Microbial” and “Targeted Internal Control”) from those that are not targeted (“Untargeted”), are low complexity (“Low Complexity”), cannot be unambiguously assigned to one category (“Ambiguous”), or cannot be classified with confidence (“Unclassified”).
Pango lineage
The most likely Pango (phylogenetic assignment of named global outbreak) lineage is assigned to the majority consensus SARS-CoV-2 genome sequence using pangolin 4.3.1 (Áine O'Toole & Emily Scher et al. 2021 Virus Evolution DOI:10.1093/ve/veab064).
Limitations
Non-detected results do not rule out the presence of viruses and AMR markers. Contamination is possible during specimen collection, transport, and processing. Closely related viruses may be misidentified based on sequence homology to viruses present in the database. The identification of cDNA or DNA sequences from a virus does not confirm that the identified virus is causing symptoms, is viable, or is infectious. Recombinant viral strains may not be reported or may be reported as one or more individual viruses. Should one or more individual viruses be reported for a recombinant viral strain, antiviral resistance results may be inaccurate. In viral strains containing insertion-deletion mutations (indels), there is a risk of false positive or false negative results for other resistance mutations within a region of 100 nucleotides around the indel.
Limitations
Information provided by DRAGEN Microbial Enrichment Plus is based on scientific knowledge and has been curated; however, scientific knowledge evolves and reported information may not always be complete and/or correct. Results should be interpreted in the context of all available information. Other sources of data may be required for confirmation.
Read QC
Low-quality bases are trimmed from the ends of each read. After trimming, the read is discarded if fewer than 50% of its bases have a quality score greater or equal to q20, the read is shorter than 32 bp, or the read has 5 or more ambiguous bases. It is assumed that appropriate adapter trimming has already been performed.
Optional
X
X
X
X
X
X
Dehosting
Human read removal using the DRAGEN Kmer Classifier
X
X
X
X
X
X
Sample QC
Sample composition and enrichment factor calculations
Internal control required to calculate the enrichment factor
X
X
X
Microorganism classification
Pre-alignment filtering step
Configurable sensitivity
X
X
X
Microorganism detection
Reference alignment, consensus sequence generation, variant calling
X
X
X
X
X
X
Microorganism quantification
Absolute copies/mL calculation
Quantitative internal control and concentration required
X
X
X
Microorganism reporting thresholds
Proprietary algorithms or user-defined reporting logic
X
X
X
X
X
Bacterial AMR marker analysis
Nucleotide and protein alignment, consensus sequence generation, variant calling and annotation
X
X
Viral AMR marker analysis
Variant calling and annotation
X
X
X
X
Viral clade and lineage prediction
Pangolin, Nextclade
X
X
X
X
Result filters
User-specified filters applied
X
X
X
X
X
Reporting - Analysis level
XLSX, HTML, ZIP
X
X
X
X
X
X
Reporting - Sample level
JSON, HTML, FASTA (consensus sequences), VCF (viral variants)
X
X
X
X
X
X
Component versions
Test type, version:
RPIP 6.5.1
UPIP 8.6.0
RVOP 2.7.0
VSP 2.7.0
VSPv2 2.7.0
Custom 1.0.0
Analysis Pipeline version: 6.3.12
DRAGEN version: 4.3.11
Third-party versions
Pangolin 4.3.1 (Pangolin database PUSHER version 1.27)
Nextclade 3.5.0
SnpEff 5.1
ResFinder (version 2.2.1)
NCBI Reference Gene Catalog (version 2023-09-26.1)
EUCAST expert rules on indicator agents (2019-2023)
CLSI Performance Standards for Antimicrobial Susceptibility Testing (M100 34th Edition)
Comprehensive Antibiotic Research Database (CARD, version 3.2.8)
Comprehensive Antibiotic Research Database Prevalence Data (CARD Prevalence, version 4.0.1)
World Health Organization (WHO) Influenza virus neuraminidase inhibitor (NAI) and polymerase acidic protein inhibitor (PAI) Reduced Susceptibility Marker Tables (07 March 2023 version)
Key updates
Various bug fixes (see below)
Tiered reporting added for Norovirus (GI, GII, GIV, GVIII, GIX) and Dengue virus (type 1, type 2, type 3, type 4)
Tiered reporting suppressed for below subtype resolution of Influenza A virus subtypes H1N1 and H3N2
Nextclade datasets added for Measles virus (MV) and Dengue virus (DENV) clade assignment
Reference genomes added for Monkeypox virus (MPV) Clade 1b
Additional database curation
Known issues
When reading Biosamples from a Project, Fastq files for Biosamples sharing the same sample name prefix before the first underscore are merged. For example, Fastq files for Biosamples PREFIX_001 and PREFIX_002 will be merged and reported as a single PREFIX sample. To avoid this error, ensure that sample names are unique before the first underscore, replace underscores with a hyphen, or provide Biosample input from a list
Coverage results for SARS-CoV-2 are slightly (<1%) over-estimated, which may result in coverage >100% due to an error accounting for masked polyA-tail bases
Viral genome consensus sequence bases without aligned read support are indicated by "X" bases rather than "N" bases for RPIP viruses except SARS-CoV-2 and Influenza viruses
Variant annotation information for Influenza A and B viruses, including antiviral resistance prediction results, may not be populated when below threshold reporting is enabled and/or a user-defined microorganism reporting file is specified that does not include all members of the Influenza A and B virus tiered reporting groups. If viral variant annotation is of interest for Influenza A and B viruses, default microorganism reporting options are recommended
Known limitations
When providing Biosample input from a list, 99 associated Fastq files is the maximum allowed per analysis. There is no Fastq file limitation when reading Biosamples from a Project
In strains containing insertion-deletion mutations (indels), there is a risk of false positive or false negative results for other resistance mutations within a region of 100 nucleotides around the indel
In strains containing long insertion-deletion mutations (indels), there is a risk of false negative results for two large vAMR-associated deletion mutations (RPIP, VSPv2) and one large bAMR-associated insertion mutation (RPIP). Long indels may be incorrectly reported as other variant types, such as frameshift mutations
Small differences in SARS-CoV-2 and Influenza virus results may be observed between repeat analyses
Bug fixes
Nextclade parsing errors for some samples
Custom reference sequence analysis not functional in non-US regions
User-defined microorganism reporting feature not reporting microorganisms that belong to a tiered reporting group when “prediction_logic” column set to “default”
RPKM and absolute quantity metrics inaccurate when read QC disabled
SHV beta-lactamase AMR markers incorrectly reported as carbapenemases based on a known curation error in CARD version 3.2.8
Reads duplicated for samples with a single FASTQ file
Empty FASTQ files abort analysis
Pangolin not run on all samples with SARS-CoV-2 detected
Viral genome coverage plots not rendered for segmented viruses when all segments not detected
Description information missing for some viral genome accessions
Initial release.
Component versions
Test type, version:
RPIP 6.3.0
UPIP 8.4.0
RVOP 2.3.0
VSP 2.3.0
VSPv2 2.3.0
Custom 1.0.0
Analysis Pipeline version: 6.3.12
DRAGEN version: 4.3.6
Third-party versions
Pangolin 4.3.1 (Pangolin data 1.27)
Nextclade 3.5.0
SnpEff 5.1
ResFinder (version 2.2.1)
NCBI Reference Gene Catalog (version 2023-09-26.1)
EUCAST expert rules on indicator agents (2019-2023)
CLSI Performance Standards for Antimicrobial Susceptibility Testing (M100 34th Edition)
Comprehensive Antibiotic Research Database (CARD, version 3.2.8)
Comprehensive Antibiotic Research Database Prevalence Data (CARD Prevalence, version 4.0.1)
World Health Organization (WHO) Influenza virus neuraminidase inhibitor (NAI) and polymerase acidic protein inhibitor (PAI) Reduced Susceptibility Marker Tables (07 March 2023 version)
Key updates
Updated and expanded microorganism and bacterial AMR marker databases
Updated and expanded Influenza virus typing capability and antiviral resistance (AVR) reporting
User-defined microorganism reporting list and reporting thresholds
Below threshold reporting for microorganisms and/or AMR markers
Custom reference sequence analysis
Known issues
When reading Biosamples from a Project, Fastq files for Biosamples sharing the same sample name prefix before the first underscore are merged. For example, Fastq files for Biosamples PREFIX_001 and PREFIX_002 will be merged and reported as a single PREFIX sample. To avoid this error, ensure that sample names are unique before the first underscore, replace underscores with a hyphen, or provide Biosample input from a list
Reads are duplicated for samples with a single FASTQ file
Empty FASTQ files will abort analysis
Nextclade may encounter a parsing error for some samples. If an analysis fails, try re-running the analysis with Nextclade disabled
Pangolin may not be run on all samples with SARS-COV-2 detected
Custom reference sequence analysis is not functional in non-US regions
The user-defined microorganism reporting feature does not report microorganisms that belong to a tiered reporting group when the “prediction_logic” column is set to “default”. See the User Guide for further information about microorganism tiered reporting
RPKM and absolute quantity metrics are inaccurate when read QC is disabled
SHV beta-lactamase AMR markers are incorrectly reported as carbapenemases based on a known curation error in CARD version 3.2.8
Coverage results for SARS-CoV-2 are slightly (<1%) over-estimated, which may result in coverage >100% due to an error accounting for masked polyA-tail bases
Viral genome consensus sequence bases without aligned read support are indicated by "X" bases rather than "N" bases for RPIP viruses except SARS-CoV-2 and Influenza viruses
Variant annotation information for Influenza A and B viruses, including antiviral resistance prediction results, may not be populated when below threshold reporting is enabled and/or a user-defined microorganism reporting file is specified that does not include all members of the Influenza A and B virus tiered reporting groups. If viral variant annotation is of interest for Influenza A and B viruses, default microorganism reporting options are recommended
Known limitations
When providing Biosample input from a list, 99 associated Fastq files is the maximum allowed per analysis. There is no Fastq file limitation when reading Biosamples from a Project
Small differences in results may be observed between repeat analyses
In strains containing insertion-deletion mutations (indels), there is a risk of false positive or false negative results for other resistance mutations within a region of 100 nucleotides around the indel
In strains containing long insertion-deletion mutations (indels), there is a risk of false negative results for two large vAMR-associated deletion mutations (RPIP, VSPv2) and one large bAMR-associated insertion mutation (RPIP). Long indels may be incorrectly reported as other variant types, such as frameshift mutations
The RPIP, VSPv2, VSPv1, and RVOP Data Analysis solutions can report Influenza A virus subtypes H1N1 and H3N2 to a below-subtype resolution. Multiple results for H1N1 and/or H3N2 may be reported concurrently, particularly in samples that contain a mixture of Influenza A virus subtypes
Viral genome coverage plots are not rendered for segmented viruses when all segments are not detected
Description information is missing for some viral genome accessions
AMR
antimicrobial resistance
mL
milliliter
NAI
neuraminidase inhibitor
NGS
next-generation sequencing
PAI
polymerase acidic endonuclease inhibitor
pangolin
phylogenetic assignment of named global outbreak lineages
RPKM
targeted Reads mapped Per Kilobase of targeted sequence per Million quality-filtered reads
VSP
Viral Surveillance Panel
RUO
For Research Use Only. Not for use in diagnostic procedures.
URL
See https://www.illumina.com/ for additional information.
Quantification
VSP data analysis using DRAGEN Microbial Enrichment Plus detects 149 viruses and 238 AMR markers based on target enriched next-generation sequencing (NGS) of viral DNA and cDNA sequences. Sequencing data are interpreted by the DRAGEN software platform and viruses that pass detection thresholds are reported. Relative abundance is expressed as proportion of RPKM values.
AMR
This test detects 238 antimicrobial resistance (AMR) markers associated with resistance to Influenza virus neuraminidase inhibitor (NAI) and polymerase acidic protein inhibitor (PAI) in Influenza A virus (H1N1pdm09), Influenza A virus (H1N1), Influenza A virus (H5N1), Influenza A virus (H3N2), Influenza A virus (H3N2; swine-like), Influenza A virus (H7N9), and Influenza B virus. AMR markers and drug associations are based on the World Health Organization (WHO) Influenza virus NAI and PAI Reduced Susceptibility Marker Tables (07 March 2023 version). Detection of an AMR marker is reported if the marker passes a minimum detection threshold and if the Influenza virus associated with the marker is also detected. Reported AMR markers have been associated with antimicrobial resistance but may not always indicate phenotypic resistance. Failure to detect AMR variants does not always indicate phenotypic susceptibility. Results should be interpreted in the context of all available information.
AMR
Mutations connected with a '+' form an epistatic group. Epistatic groups are two or more mutations that need to be present concurrently to confer the associated resistance.
Pango lineage
The most likely Pango (phylogenetic assignment of named global outbreak) lineage is assigned to the majority consensus SARS-CoV-2 genome sequence using pangolin 4.3.1 (Áine O'Toole & Emily Scher et al. 2021 Virus Evolution DOI:10.1093/ve/veab064).
Limitations
Non-detected results do not rule out the presence of viruses and AMR markers. Contamination is possible during specimen collection, transport, and processing. Closely related viruses may be misidentified based on sequence homology to viruses present in the database. The identification of cDNA or DNA sequences from a virus does not confirm that the identified virus is causing symptoms, is viable, or is infectious. Recombinant viral strains may not be reported or may be reported as one or more individual viruses. Should one or more individual viruses be reported for a recombinant viral strain, antiviral resistance results may be inaccurate. In viral strains containing insertion-deletion mutations (indels), there is a risk of false positive or false negative results for other resistance mutations within a region of 100 nucleotides around the indel.
Limitations
Information provided by DRAGEN Microbial Enrichment Plus is based on scientific knowledge and has been curated; however, scientific knowledge evolves and reported information may not always be complete and/or correct. Results should be interpreted in the context of all available information. Other sources of data may be required for confirmation.
Application note:
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Genomics Research Hub (GRH) article:
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UPIP ID Week Scientific Poster:
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