How to use a custom virus primer BED file with the DRAGEN COVID Lineage app on BaseSpace

The following reviews how to use a custom virus primer BED file with the DRAGEN COVID Lineage app on BaseSpace Sequence Hub.

When launching the DRAGEN COVID Lineage app, the app prompts the selection of a Virus Primers BED file. It is recommended that users select a BED file as input when analyzing samples prepared using an amplicon-based library prep kit. Reads that start in the defined primer binding sites and have the same orientation are presumed to be of possible primer origin, and the portion of the read that overlaps the primer binding site will be hard-clipped and not considered for variant calling. In addition, any portion of the reference genome that is not tiled by an amplicon defined by this primer set will be masked in the consensus sequence.

The app currently provides built-in support for ARTIC V3, V4, and V4.1 (experimental) Primers. Virus primers can be disabled by selecting None; this is applicable for samples prepared using non-amplicon library types, such as enrichment.

The app also supports the use of a custom virus primer BED file. This BED file must be formatted as per the ARTIC Primer Scheme, defined here. The following are key features that the BED file has in order to be compatible with the DRAGEN COVID Lineage app.

• The primer BED must have a maximum of 6 columns.

  
• The primer reference sequence mentioned in column 1 must match the reference used for analysis. By default, the DRAGEN COVID Lineage app uses NC_045512.2, but a custom reference can be used if needed.

MN908947.3 is another reference that is commonly specified in non-ARTIC primer BED files. This reference has the same sequence as NC_045512.2 and hence, “MN908947.3” can simply be replaced by “NC_045512.2” in these BED files (and the app can be launched without selecting a custom reference).

• The coordinates in columns 2, and 3 need to be 0-based; the reference sequence position counting starts at 0, and chromEnd is not included in the primer sequence.

• The primer name (column 4) should contain a ‘_LEFT’ or a ‘_RIGHT’ tag and can, optionally, contain an ‘_alt’ tag as well. The tag can be located anywhere within the primer name. Each forward primer must have a reverse primer with a matching base name (primer name after removing the tags) within the same primer pool.

• The BED file must be sorted in ascending order, based on the coordinates in column 2 (chromStart). Though the ARTIC primer scheme document does not mention this as a requirement, the DRAGEN COVID Lineage app will abort if an unsorted BED file is used for analysis.

Examples of the primer BED can be found on the ARTIC network's GitHub page (file named SARS-CoV-2.primer.bed).

For any feedback or questions regarding this article (Illumina Knowledge Article #6251), contact Illumina Technical Support [e-mail].