❓Frequently Asked Questions (FAQ)

Q: Which infectious disease and microbiology enrichment panels are compatible with analysis in DRAGEN Microbial Enrichment Plus?

A: The following enrichment panels can be analyzed using the app: VSP, VSP V2, RVOP/RVEK, RPIP, and UPIP enrichment panels. In addition, custom infectious disease and microbiology panels can also be analyzed. The Pan-Coronavirus Panel cannot be analyzed in DME+ using default settings; however, a custom coronavirus reference database can be used to analyze this panel. This application was not intended for non-infectious disease enrichment panels (such as exome) and was not intended for other types of workflows (such as shotgun metagenomics).

Q: What does it cost to analyze samples using this app?

A: There is no compute cost to run the application. A Basic BSSH account is needed, and additional storage may require iCredits.

Q: How can I verify or compare results of the DRAGEN Microbial Enrichment Plus analysis pipeline to previously used apps (such as DRAGEN Targeted Microbial)?

A: Fastq files previously run through other apps can be re-analyzed using DME+. Results from other pipelines may not be identical to DME+, most notably because of the unique databases used in DME+. To perform a direct comparison from DME+ to a different analyses pipeline, we recommend that the consensus FASTA files generated be run through NCBI Blast to assess sequence similarity.

Q: I don't see the virus/microbe I'm interested in listed in the summary of reported Microbial Organisms. Does that mean my organism is not present?

A: Not necessarily. The microbe of interest may be present in the sample, but the app may not have reported it because the alignment metrics fell below the default prediction thresholds. If you suspect this may be the case, select the "Report microorganisms and/or AMR markers that are below threshold" option. If you know a priori which organisms are expected, the "Custom panel specification" can also be used.

Q: I am using the custom analysis workflow and my analysis aborted or shows an error, why?

A: While there may be quite a few causes for the analysis to fail, one of the most common causes is that the custom database was not formatted correctly. Below are requirements for the "Custom reference FASTA for consensus generation":

• Do not use Spaces in the file name; instead use an underscore "_"

• Do not exceed 25 characters in the file name

• File extension must be .fasta or .fa

• Do not exceed the file size limitation: 16GB for a single file

• Do not have duplicate entries

• If providing a Custom Reference BED and/or Custom PCR Primer Definitions in BED format, the names in the first column of the BED file (chrom) must match the names that appear in the FASTA (text after > and before the first whitespace character).

Q: I am using the "User-defined specification" option for UPIP, RPIP, VSP, VSP V2, or RVOP. I am not seeing the microorganisms I expect to be there, OR I am seeing microorganisms that I do not want to see.

A: Ensure that the correct file with the intended microorganisms was uploaded and used. We recommend saving the updated microorganism file with a new name. Do not add any new columns or delete any columns form the excel template. Similarly, do not change or remove any items in the Header row, but rows with microorganism names that you are not interested in can be deleted.

Q: What does "Read classification sensitivity" mean in the settings for VSP, VSP V2, or RVOP/RVEK?

A: If analyzing with VSP, VSP V2 or RVOP panels, the default read classification sensitivity is set to 5. This setting is used as a pre-alignment filtering step. If less than 5 reads classify to the set of reference sequences belonging to a given organism, that organism will not be reported. If 5 or more reads classify to the set of reference sequences belonging to a given organism, analysis will proceed to a read alignment workflow, and alignment-based thresholds will be used to determine whether that organism is reported. Therefore, even if there are >5 reads mapping to a viral group, the final analyses will not report that virus if the aligmment-based thresholds are not met. 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.

Q: Where do I upload my reference fasta and/or BED file for a custom analysis?

A: Upload these files to a basespace project before launching the DME+ app; you will then be able to select these files in the "Select Dataset File(s)" browser in the app. Please see general guidelines for how to upload data to BaseSpace. If you continue having issues, reach out to [e-mail]

Q: Can I analyze the Pan-Cov panel here?

A: The only infectious disease and microbiology panel that is not pre-set in this application is the Pan-CoV enrichment panel. To use the Pan-CoV enrichment panel with the DME+ app, select the "Custom panel" under the "Enrichment Panel" drop-down list and use a custom reference database. Otherwise, we recomend using the DRAGEN Targeted Microbial Enrichment BaseSpace app.