# Pipeline Logic

<figure><img src="/files/QR2dUNaR15SrUNRYxz9l" alt=""><figcaption><p>Control and data flow diagram for the DRAGEN Microbial Amplicon app. Not all steps shown may be run depending on user inputs and pipeline outcome</p></figcaption></figure>

## Steps

| Step                                        | Module/Script | Run                                                                      |
| ------------------------------------------- | ------------- | ------------------------------------------------------------------------ |
| QC                                          | trimmomatic   | Always                                                                   |
| Primer trimming (on FASTQ)                  | trimmomatic   | If assembly is to run                                                    |
| Remove off-target reads                     | DRAGEN        | If checked in Input Form                                                 |
| Assembly                                    | MEGAHIT       | If reference FASTA and BED files imply more than one genome as reference |
| Contig clustering                           | CD-HIT        | If assembly ran                                                          |
| Reference selection                         | custom script | If assembly ran, otherwise input reference database is used as is        |
| Map/Align                                   | DRAGEN        | If at least one reference sequence is generated                          |
| Post-facto primer trimming (on BAM)         | custom script | If Map/Align ran and primer set exists                                   |
| Sample filtering based on amplicon coverage | custom script | If Map/Align ran and primer set exists                                   |
| Variant calling                             | DRAGEN        | If Map/Align ran and sample passed filter above                          |
| Consensus sequence generation               | custom script | If Map/Align ran and sample passed filter above                          |

## Outcomes

<table><thead><tr><th width="328">Status</th><th width="92">Level</th><th>Outcome</th></tr></thead><tbody><tr><td>Completed successfully</td><td>Pipeline</td><td>Exit with all applicable output files</td></tr><tr><td>Custom files are not formatted correctly</td><td>Pipeline</td><td>Exit early with error</td></tr><tr><td>No remaining reads after preprocessing</td><td>Sample</td><td>Exit early with a report of read counts</td></tr><tr><td>No contig generated</td><td>Sample</td><td>Exit early with a report of read counts</td></tr><tr><td>No reference found after assembly</td><td>Sample</td><td>Exit early with a report of read counts and contig FASTA</td></tr><tr><td>None of the primers provided in custom primer definition file align to selected reference sequences</td><td>Sample</td><td>Skip post-factor primer trimming and sample filtering based on amplicon coverage for this sample</td></tr><tr><td>Insufficient amplicon coverage</td><td>Sample</td><td>Exit early before variant calling and consensus sequence generation</td></tr></tbody></table>


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