DNA Barcoding workflow

The DNA Barcoding tutorial uses example data found on the ONTOLOGY demo data page. The data are Australian insects collected by Paul Hebert via Malaise traps in December 2024. Two ONTOLOGY plates—UMIs 1 and 2—were prepared and sequenced on an Oxford Nanopore Technologies’ Flongle Flow Cell on a MinION Mk-1d in September 2025. A single .fastq file, containing 17,912 reads, from the run is included for computational efficiency; the full run contained nearly 1M reads and takes 20× longer.

Because the ‘Implement’ stage is hands-on, this tutorial does not go into high level detail for this stage.

The advanced user modules simplify PREPARE, skip IMPLEMENT, and include the full ANALYZE. Therefore, the tutorials for the advanced user modules only focus on PREPARE.

Note: With all modes, please refrain from naming samples with terminology used frequently for controls. These include any capitalization of ‘pos, ‘positive’, ‘’neg’, ‘’negative’, & ‘control’

Standard mode (with ONTOLOGY kit)

Open ONTOLOGY and select DNA Barcoding

  1. PREPARE

    1. Select New Run

    2. For Run name, you can enter anything you’d like, but we suggest DBC_Full_Tutorial

    3. For Batch ID, enter exactly OAT001, and leave the marker as COI and the primer as AR01.

    4. Click Initialize.

    e. The next three screens indicate the current plate being prepared and provide workflow and specimen information. Click the forward arrow (->) several times to proceed through the workflow until you arrive at the Array screen. Here you will see an option to Download a prepared Metadata Template file that may be edited and re-uploaded on future uses. Please take a moment to download and review the file. For now, you do not need to change this file, but it may be used to compare with the completed Metadata files we have provided (metadata_sheet_for_01.xlsx), which will be used for upload on the next screen.

    REQUIREMENTS FOR THE METADATA SHEET: Upon using your own data, you will need to edit the metadata sheet yourself. After downloading the template metadata sheet. Required Fields:

    • Sample ID: Do not make any changes to data in the Sample ID field.

    • Custom ID: Provide a memorable ‘working name’ to each cell in the Custom ID field. This may match the naming convention you have used for sample collection and organization.

    The only requirement is that these must be unique identifiers for each row (e.g., 1,2,3…). * Kingdom/Phylum/Class/Order fields: Change these fields as needed to represent what is known about your samples. For taxonomic inference, all fields up to Order must be filled in. * Country: Enter country name only. * All fields: For each plate, Well H12 is designated as the Negative Control well. The Negative Control must remain blank for all fields, excluding Sample ID.

    Optional fields: We highly recommend that you fill out the Collectors, Collection Date, Latitude, and Longitude fields, though they are not required for ONTOLOGY to run. Please refer to the provided metadata_sheet_for_01.xlsx for suggested formatting.

    1. Click the forward arrow once more to uploade prepared metadata file.

      • Select Choose File

      • Navigate to ‘DNAbarcode_standard_tutorial/metadata’

      • Select metadata_sheet_for_01.xlsx

      • Confirm all details are correct (95 specimens; 1 negative control; 95 specimens with complete data) and then click the forward arrow.

    2. The next screen provides a downloadable Sampling Protocol, which is optional for review. Continue past this screen, and click the forward arrow until you arrive at Label. Here we will upload an array image. This is optional but encouraged as it allows for improved error checking.

      • Select Upload Array Image

      • Navigate to ‘DNAbarcode_standard_tutorial/array_images’

      • Select array_01.jpg

      • Note: this image is from a separate project but was chosen because it contains pinned insects; most users would not upload an array photo of a microplate.

    3. Select the forward arrow to proceed to PHOTOGRAPH

      • Select Upload photographs

      • Navigate to ‘DNAbarcode_standard_tutorial/specimen_images’

      • Select the folder, ‘plate1’, then click Open

      • Verify the number of images is correct (95 images uploaded), then click the forward arrow

    4. Review the Plate Summary page. The user can use the ‘Edit’ buttons to make changes to the metadata, if desired.

    5. Select “Yes, add another plate (repeat Prepare Specimens)”

    6. Repeat Steps f to i above, but choose metadata_sheet_for_02.xlsx, array_02.jpg, and the images in the ‘plate2’ older.

    7. Review the summary sheet—all wells on both plates should be green—and then select No, all specimens processed.

  2. IMPLEMENT

    1. Click through all arrows of IMPLEMENT to explore the protocols.

    2. Explore the interactive calculators at SEQUENCING > LIBRARY PREP and SEQUENCING > FLOW CELL

    3. Proceed to ANALYZE

  3. ANALYZE

    1. Upload the .fastq file (or files, if using the full run sequences)

      • Make sure that the Run the complete analysis workflow is selected.

      • Select Choose Folder

      • Navigate to ‘DNAbarcode_standard_tutorial/fastq_file’ and ‘open’ that folder

      • Verify that the number of .fastq files is correct (1) and the number of reads is 17,912 then click forward arrow

    2. In Select Reference Library:

      • If the ‘Current Reference Library’ is blank, download BOLDistilled via the ‘Download BOLDistilled’ button.

      • Once the BOLDistilled reference library autopopulates the Reference Library field, you can ‘Proceed to Analysis’.

    3. At the Bioinformatic Analysis stage, select Start Analysis; the analysis should complete in under 10 minutes on most computers. After analysis has completed, click the forward arrow.

    4. Explore the various summary metrics and graphs on the next page.

      • You can also find file versions by clicking the ONTOLOGY Workspace button on the right side of the screen.

      • Select the folder with the batch name assigned (e.g., OAT001), then ‘{batch name}’ -> ‘analyze’, and you will find all saved ONTOLOGY output files.

    5. On the next page, type your email and the desired BOLD project code and then click Submit data package (note, the tutorial data will not go to BOLD)

    6. The RUN SUMMARY page summarises the details of the completed run.

Advanced user (with ONTOLOGY kit)

Open ONTOLOGY and select DNA Barcoding. [Do not select New Run yet]. Then, select the Switch to Advanced Mode button (S <–> A image) on the right side to launch the advanced used module.

  1. PREPARE

    1. Click the forward (–>) arrow until the RUN SETUP screen. For Run name, you can enter anything you’d like, but we suggest ‘DBC_Advanced_Tutorial’

    2. In the Kit Info fields, enter OAT001 as the batch ID and leave the marker as COI and the primer as AR01.

    3. Check boxes 1 and 2 for ‘Select Plate(s)’ to indicate number of plates that are being assessed.

    4. Click ‘Initialize’.

    e. Click ‘Download Blank’ next to Metadata. Please take a moment to download and review the file. For now, you do not need to change this file, but it may be used to compare with the completed Metadata files we have provided (metadata_sheet_for_all.xlsx), which will be used for upload on the next screen.

    REQUIREMENTS FOR THE METADATA SHEET: Upon using your own data, you will need to edit the metadata sheet yourself. After downloading the template metadata sheet. Required Fields:

    • Sample ID: Do not make any changes to data in the Sample ID field.

    • Custom ID: Provide a memorable ‘working name’ to each cell in the Custom ID field. This may match the naming convention you have used for sample collection and organization.

    The only requirement is that these must be unique identifiers for each row (e.g., 1,2,3…). * Kingdom/Phylum/Class/Order fields: Change these fields as needed to represent what is known about your samples. For taxonomic inference, all fields up to Order must be filled in. * Country Enter country name only. * All fields: For each plate, Well H12 is designated as the Negative Control well. The Negative Control must remain blank for all fields, excluding Sample ID.

    Optional fields: We highly recommend that you fill out the Collectors, Collection Date, Latitude, and Longitude fields, though they are not required for ONTOLOGY to run. Please refer to the provided metadata_sheet_for_all.xlsx for suggested formatting.

    1. Click the forward arrow once more.

    2. Select ‘Upload Metadata’ and then select the ‘DNAbarcode_advanced_tutorial/metadata_sheet_for_all.xlsx’ file we provided (not the ONTOLOGY-generated template you downloaded)

    3. Select specimen images, then select the ‘DNAbarcode_advanced_tutorial/specimen_images’ folder

    4. Select ‘DNAbarcode_advanced_tutorial/array images’, then select all files in the folder

    5. To progress to Analyze:

      • Click the forward arrow

      • Then review the Run Summary.

      • Once your review is complete, click the forward arrow to proceed to Analyze

  2. ANALYZE

Analyze is identical to the full walkthrough; see Standard Mode

Custom mode

Open ONTOLOGY and select DNA Barcoding. Then, select the Switch to Advanced Mode button (S <–> A image) on the right side to launch the advanced used module. Because Custom Mode is similar to Kit mode, we encourage you to use your own data to get the most out of this tutorial. However, you may also use the data provided in ‘DNAbarcode_custom_tutorial’ to simulate running your own data.

  1. PREPARE

    1. Click the forward (–>) arrow until the RUN SETUP screen.

      • Toggle the switch Switch between Kit Mode and Custom Mode (top right).

      • You are now in Custom Mode.

    2. For Run name, you can enter anything you’d like, but we suggest ‘DBC_Custom_Tutorial’.

    3. Click ‘Initialize’.

    4. For Mapping File, click Download Template.

    5. Fill in Mapping Template File.

      • Note that you MUST provide your own UMI and primer sequences in custom mode.

      • Example row: Plate ID = 1 (for 96 samples or less), Well ID = B01, Sample ID = Study1_Sample2, Primer Set Name = AR01.

      • Note: Plate IDs must only be a number, e.g., 2 or 24.

    6. Upload Mapping File. Then, for Metadata, click ‘Download Blank’.

    7. Fill in the Metadata file, which should correspond with the Mapping file. NOTE: do not change the Sample ID or Custom ID fields. The Kingdom/Phylum/Class/Order fields are required for taxonomic inference.

    8. Upload Metadata File.

    9. Click the writing icon next to Bioinformatics Parameters.

    10. [Advised] Download Bulk Input file, check for correctness, make any necessary edits, and re-upload using Upload Bulk Input file button.

      • Parameters can also be edited directly in the ONTOLOGY window.

      • A guide to the parameters is available in the PREPARE document of the ONTOLOGY tutorial.

  2. ANALYZE

Analyze is identical to the full walkthrough; see Standard Mode