❓Frequently Asked Questions (FAQ)

General

Q: What does it cost to analyze samples using the DRAGEN 16S Plus app?

A: The compute cost to run the DRAGEN 16S Plus app is 2.19 iCredits/hr. A Basic Basespace Sequence Hub (BSSH) user account is also required to access the DRAGEN 16S Plus app. However, there is no subscription cost for a Basic BSSH account. A Basic BSSH account provides 1 TB of free storage. Additional storage may require iCredits.

Q: Where do I upload my custom database FASTA file to use with the DRAGEN 16S Plus app?

A: Upload these files to a BSSH project before launching the DRAGEN 16S Plus app. It will then be possible 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 and reach out to [email protected] with any unresolved upload issues.

Analysis Options & Settings

Q: What is the default "Refseq-RDP-v1" reference database provided with the DRAGEN 16S Plus app?

A: The Refseq-RDP-v1 database contains 14,676 bacterial & 660 archaeal full-length 16S rRNA gene sequences. It was compiled in 14/05/2018 from predominantly the NCBI RefSeq 16S rRNA database and was supplemented with extra sequences from the RDP database. The same 16S sequence content was used to build the default 16S database provided with the 16S Metagenomics v1.1.3 app (RefSeq RDP 16S v3 May 2018 DADA2 32 bp).

Citation: Ali Alishum. (2019). DADA2 formatted 16S rRNA gene sequences for both bacteria & archaea (Version 1) [Data set]. Zenodo. https://doi.org/10.5281/zenodo.2541239

Q: Can I use the "Custom database specification" option in the DRAGEN 16S Plus app to analyze ITS, 18S, and/or 16S data using a different database?

A: Yes. A custom database FASTA file containing up to 500 million basepairs of reference sequence may be specified using the exact FASTA header format defined below. In the FASTA file, the SequenceID should not contain any spaces. All sequences must have seven canonical taxonomic rank prefixes specified: k__;p__;c__;o__;f__;g__;s__. However these can all be left blank except for (k)ingdom and (s)pecies designations, which are required.

>SequenceID_001:k__Fungi;p__Glomeromycota;c__Glomeromycetes;o__Glomerales;f__Glomeraceae;g__;s__uncultured_Glomus

See Custom reference FASTA and BED files for further details.

Q: What read QC (Quality Control) is performed by the DRAGEN 16S Plus app?

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. For paired-end data, both read1 and read2 must pass QC filtering for the read pair to be used for analysis. It is assumed that appropriate adapter and primer trimming has already been performed.

Q: What does the "Read count threshold" mean in the settings for the DRAGEN 16S Plus app?

A: The read count threshold is a reporting threshold representing the number of reads (or read pairs for paired-end data) that must be classified to a taxonomic identifier for the taxonomic identifier to be included in summary reports. It is an integer with a valid range of 0 to 1000, inclusive. The default value is 0, meaning that no filtering is applied to results and all taxa with classified reads are reported.

Reporting

Q. Are low read count taxa actually in the sample or are they artifacts?

A: Low read count results could represent true low positives or non-specific results, contamination, etc. If maximum confidence is required for a given use case, a higher read count reporting threshold can be specified.

Results & Output Files

Q: How can I verify or compare results of the DRAGEN 16S Plus app to previously used apps (such as 16S Metagenomics)?

A: FASTQ files previously run through other apps can be re-analyzed using the DRAGEN 16S Plus app. Results from other apps may not be identical to results from the DRAGEN 16S Plus app, most notably because of algorithm and/or database differences.