🌀How to set up and run an analysis

1. Launch the DRAGEN Microbial Enrichment Plus BaseSpace app, which can be found in the "Dragen" and "Infectious Disease + Microbiology" app collections.

2. Enter a name for the Analysis.

3. 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.

4. 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 Custom reference FASTA and BED files for further details.

5. 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.

   • 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 Microorganism Reporting File format for further details.

6. 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

1. 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.

2. 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

3. 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)

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

5. 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.

6. Select the Project where the Analysis Output should be saved.