Add a custom reference

To create a custom reference, upload a nucleotide sequences FASTA file and an optional polymorphic regions BED file. The FASTA format file contains the context sequence. If there are known polymorphic regions in the FASTA reference sequence, such as SNPs and INDELs, you can upload an optional BED file that contains regions of high variation over which to avoid placing primers for designs against the custom reference. For more information about how the pipeline avoids variant regions during the design process, see Basic and biological filtering.

IMPORTANT! Custom genome references are compatible only with Ion AmpliSeq™ DNA designs.

  1. In the navigation bar, click Genomes.
  2. In the Design References screen, click Add reference.
  3. In the Add a custom reference screen, complete the required information.

    Field

    Description

    Reference name[1]

    Enter a unique name for the sequence to be used as a reference for your design. The name can be 3–32 characters in length. The allowed characters are US‑ASCII letters, numbers, spaces, asterisks (*), dashes (-), and underscores (_).

    Associated organism for primer specificity check1

    Select an associated organism for primer specificity check to improve primer specificity to your custom reference. Primary specificity is improved by favoring primers with few optimal binding sites in the consensus sequence.

    Primer specificity check refers to the process of identifying potential primer mispriming events. Primers with high number of potential mispriming events are avoided in panel designs. If you select an associated organism, designs for the custom reference will perform primer specificity check against the preloaded genome sequence of the selected organism. If you select None, primer specificity check will be performed against the custom reference.

    Reference source (Recommended)

    The name of the database or source of the reference nucleotide sequence.

    Reference description

    Add any notes regarding the custom reference sequence. The description can be up to 2,000 characters in length.

    Reference sequences (FASTA) file1

    Upload one or more reference sequences FASTA files, or enter FASTA data in a text area. For more information on FASTA format requirements, see FASTA format requirements.

    • To upload a file, click Select file, select the FASTA file of interest from your local storage, then click Open.

      Note: The maximum upload file size is 2.0 GB. The limit can be extended up to 4.0 GB on request.

    • To enter or copy/paste FASTA data, click Enter FASTA data in a text area instead, then enter the reference sequence data in the provided field.

    Reference short name for Torrent Server1

    Enter a name for your reference to be used by Torrent Suite™ Software. The name should be 1–30 characters in length. The allowed characters are lowercase US-ASCII letters, numbers, and underscores (_). For more information on importing custom references into Torrent Suite™ Software, see the Torrent Suite™ Software Help for your version of the software.

    Known polymorphism (BED) file

    A BED format file that indicates regions of the sequences in the custom reference FASTA file with high polymorphism (for example, SNPs, INDELs, or other variations). Ion AmpliSeq™ Designer minimizes primer overlap with these regions. This file is optional. For specifications on creating and formatting BED files for uploading, see Known polymorphism BED file requirements.

    To upload a file, click Select file, select the BED file from your local storage, then click Open.
    1 Required field
  4. When the file upload is complete, click Save.

The new custom reference is added to the list of references in the Custom References tab.

To confirm the addition of a new custom reference, click Genomes in the navigation bar, then in the Design References screen, click the Custom References tab. For more information, see View available reference genomes.

Figure 1. Custom References tab

  1. Edit custom reference details.

  2. View custom reference details.

After a new custom reference is added, you can perform the following actions.

Action

Description

View reference details

In the Custom References tab, in the Name column, click the link in the row of a custom reference of interest. See callout 2 in Figure 1.

The Custom Reference details screen opens, displaying custom reference details, including a summary of contigs and known polymorphisms, if present. See Figure 2.

Edit a reference

  1. In the Custom References tab, click Edit in the row of a custom reference of interest (see callout 1 in Figure 1). Alternatively, in the Custom Reference details screen, click Edit (see callout 2 in Figure 2).

  2. In the Edit custom reference screen, edit the available fields as necessary, then click Save.

Download the reference file

  1. In the Custom References tab, in the Name column, click the link in the row of a custom reference of interest.

  2. In the Custom Reference details screen, click Download reference file (see callout 1 in Figure 2).

The custom reference ZIP file (Custom_Reference_<reference name>.zip) is automatically downloaded to a designated location in your local storage.

Start a new design using the reference

  1. In the Custom References tab, in the Name column, click the link in the row of a custom reference of interest.

  2. In the Custom Reference details screen, click Add design (see callout 3 in Figure 2).

The Edit your design screen opens. As you proceed through the design creation workflow, the custom reference is automatically selected as the genome for the design.
Figure 2. Custom Reference details screen

  1. Download the custom references files.

  2. Edit custom reference details.

  3. Start a new design using the custom reference.

Related Topics
Parent topic: Get started with Ion AmpliSeq™ Designer