Detect Amplicon Sequence Variants and Assign Taxonomies

The Detect Amplicon Sequence Variants and Assign Taxonomies workflow processes reads from amplicon sequencing to yield a merged multi-sample (if applicable) ASV abundance table, and subsequently assigns taxonomies to the ASVs (amplicon sequence variants).

We recommend making preliminary evaluations of the read lengths and qualities, to decide on parameter settings like read length. This can be done by running a single sample through the workflow, and taking a look at the resulting trim report section Read length before / after trimming.

The workflow requires a Trim Adapter List to remove adapters from the reads and a Taxonomic Profiling Index to assign taxonomies to ASVs. If the reads have already had adapters removed, we recommend creating a copy of the workflow (right-click the workflow in the Toolbox and select 'Open Copy of Workflow'), removing the Adapter Trim List input, and saving the modified workflow. When running the modified workflow, the Adapter Trim List can be left out to skip adapter trimming, but 'Quality trimming' and 'Automatic read-through adapter trimming' will still take place.

Launching the workflow

The Detect Amplicon Sequence Variants and Assign Taxonomies workflow is at:

        Toolbox | Template Workflows (Image workflow_group) | Microbial Workflows (Image mgm_folder_closed_flat_16_h_p) | Metagenomics (Image wma_folder_open_flat_16_n_p) | Amplicon-Based Analysis (Image otutools_open_16_n_p) | Detect Amplicon Sequence Variants and Assign Taxonomies (Image detect_amplicon_sequence_variants_16_n_p)

Launch the workflow and step through the wizard.

  1. Select the sequence list(s) containing the reads to process and click on Next.
  2. Select a Trim Adapter List corresponding to the adapters used for sequencing and click on Next (figure 3.27).
  3. Select a Taxonomic Profiling Index and click on Next (figure 3.28).
  4. The 'Configure batching' and 'Batch overview' steps can be left as is (figure 3.29), or configured as described in http://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Launching_workflows_individually_in_batches.html.
  5. Choose the trim length to use for your reads and decide whether to remove chimeras by toggling the 'Remove chimeras' box. Click on Next (figure 3.30).

    The optimal read length setting will depend on the length of your reads after trimming. We recommend that you have a look at the trim report section Read length before / after trimming if you are unsure about what value to set.

  6. Finally, select a location to save outputs to and click on Finish.

Image detect_assign_asv_primer
Figure 3.27: Wizard step for selecting the Trim Adapter List.

Image detect_assign_asv_database
Figure 3.28: Wizard step for selecting the Taxonomic Profiling Index.

Image detect_assign_asv_metadata
Figure 3.29: Optional wizard step to configure metadata for the input sequences.

Image detect_assign_asv_chimeras
Figure 3.30: Wizard step for selecting read trim length and whether to remove chimeras in the Detect Amplicon Sequence Variants tool.

Tools in the workflow and outputs generated

The Detect Amplicon Sequence Variants and Assign Taxonomies workflow consists of the below mentioned tools. See figure 3.31 for a full overview of the workflow.

Image detect_assign_asv_layout
Figure 3.31: Layout of the Detect Amplicon Sequence Variant and Assign Taxonomies workflow.