A BED-like tab-separated value (TSV) file with no header row, consisting of the following columns:

1. chrom: each sequence name as it appears in Custom Reference FASTA

2. chromStart: start position (always set to 0)

3. chromEnd: end position (sequence length)

4. genomeName: full name of the virus the sequence belongs to (e.g. Monkeypox virus clade II)

5. (optional) segmentName: how this sequence is labeled within the virus (e.g. Segment 4 (HA)). Set to 'Full' if the sequence is the full genome

Guidelines

• This file affects how sequences are labeled in the output.

• Sequence names must match those in Custom Reference FASTA. The same set of sequences must appear in both.

• 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.

Example

If primer coordinates are known (recommended)

Provide a BED file with at least 4 columns: chrom, chromStart, chromEnd, primerName. Additional columns can be included: pool, strand, sequence, but their order must be maintained.

For example, chrom, chromStart, chromEnd, primerName, pool for 5-column BED format:

And chrom, chromStart, chromEnd, primerName, pool, strand, sequence for 7-column BED format:

If primer coordinates are unknown

Option 1. One line per amplicon with 3 columns: ampliconName, forwardSequence,reverseSequence.

Option 2. One line per primer with 3 columns: primerName, sequence, pool.

Formatting rules

• General

  • All text is case sensitive.

  • Any line starting with '#' is ignored. This can be used to add a header line with column names.

In addition to the built-in options, DRAGEN Targeted Microbial supports the use of custom reference genomes and primer definitions. These files must be uploaded to a BaseSpace Project before they can be used. See https://help.basespace.illumina.com/manage-data/import-data for more information about importing files into BaseSpace. These files can be used for both Enrichment and Amplicon libraries, when choosing the 'Custom' option for 'Enrichment Panel' or 'Amplicon Primer Set', respectively. Expand the 'Custom Reference' settings block to access the options for custom files. The following controls are applicable to the specified experiment type:

Custom Enrichment Panel

• Custom Reference FASTA for Consensus Generation (required)

• Custom Reference BED (optional)

Custom Amplicon Primer Set

• Custom Reference FASTA for Consensus Generation (required)

• Custom Reference BED (optional)

• Custom PCR Primer Definitions (optional)

Custom genome references

The user may provide one or more reference genomes as the target for read alignment (and as the basis for generating consensus sequences). At a minimum, the user must provide a FASTA file containing the sequences of the reference genomes. The software will generate the required DRAGEN hash tables and other auxiliary files automatically, so there is no need to process the FASTA file with a separate app. Use the 'Custom Reference FASTA for Consensus Generation' control to select the previously-uploaded FASTA file containing the reference sequences.

Optionally, a genome definition BED file may also be provided, which tells the software more information about each sequence, such as a human-readable common name to be used in the reports. For multi-segment genomes such as Influenza, the genome definition file provides the segment name of each sequence and indicates that all the segments of a single genome belong together. Use the 'Custom Reference BED' control to select the previously-uploaded BED file containing the genome definition. See the following page for a description of the format of the genome definition file:

Custom primer sets

For amplicon experiments, the user may optionally provide a file that defines the primer sequences or locations. The primers defined in this file are used for two purposes:

1. The primer binding locations are used to trim reads, which eliminates sequence data that may be contributed by the primer sequences themselves (which we do not want) from sequence data contributed by the sample (which we do want). This is important to avoid reference bias that can depress the observed allele frequency of sequence variants in primer binding sites.

2. The primers are matched to define the boundaries of the expected amplicons resulting from the PCR reaction. The read coverage within the unique (non-overlapping) regions of these amplicons is used to determine whether or not each amplicon is reliably observed. The fraction of observed amplicons is a function of the concentration of the sample, and is used to determine whether or not sufficient material exists within the sample to reliably and accurately call variants and generate a consensus sequence. See this page for a more in-depth discussion:

Use the 'Custom PCR Primer Definitions' control to select the previously-uploaded primer definition file. The allowed formats for this file are described here:

Required custom input based on reference type for amplicon experiments