.. _docs_gp_ampliconseq:

.. spelling:word-list:: 

      Amplicon
      Amplicons
      amplicon
      amplicons
      seqs 
      metagenomic   

Amplicon Sequencing Pipeline
============================

:bdg-primary:`Version` |genpipes_version|

.. warning::

   Amplicon supports only `dada2` protocol by default. The Amplicon *QIIME* protocol is **deprecated** from *GenPipes v5.x* onward.  
   To use `QIIME`` protocol, try `an older version of GenPipes <https://genpipes.readthedocs.io/en/genpipes-v4.6.0/user_guide/pipelines/gp_ampliconseq.html>`_.

.. tab-set:: 

      .. tab-item:: Usage

         .. dropdown:: Command
            :open:

            .. code-block:: bash

                  user@machine:~$ genpipes ampliconseq [options] [--genpipes_file GENPIPES_FILE.sh]
                  user@machine:~$ bash GENPIPES_FILE.sh

         .. dropdown:: Options

            .. include:: /common/gp_design_opt.inc 
            .. include:: /common/gp_readset_opt.inc 
            .. include:: /common/gp_common_opt.inc 

         .. dropdown:: Example

            .. include::  /user_guide/pipelines/example_runs/ampliconseq.inc

            .. include:: /user_guide/pipelines/notes/scriptfile_deprecation.inc

            .. card:: Test Dataset
               :link: docs_testdatasets
               :link-type: ref

               You can download the test dataset for this pipeline :ref:`here<docs_testdatasets>`.

      .. tab-item:: Schema
         :name: ampschema    

         .. dropdown:: ``dada2``
            :open:

            .. figure:: /img/pipelines/mmd/ampliconseq.dada2.mmd.png
               :align: center
               :alt: dada2 ampseq 
               :width: 70%
               :figwidth: 95%

               Figure: Schema of DADA2 Amplicon Sequencing protocol

            .. figure:: /img/pipelines/mmd/legend.mmd.png
               :align: center
               :alt: dada2 ampseq 
               :width: 100%
               :figwidth: 75%


      .. tab-item:: Steps

         .. dropdown:: DADA2
            :open:

            .. list-table:: 
               :widths: auto
               :class: table-responsive

               * - 1.
                 - |trimmomatic16S| 
               * - 2.
                 - |merge_trimmomatic_stats16S| 
               * - 3.
                 - |flash_pass1|  
               * - 4.
                 - |ampliconLengthParser|    
               * - 5.
                 - |flash_pass2|   
               * - 6.
                 - |merge_flash_stats|     
               * - 7.
                 - |asva|    
               * - 8.
                 - |run_multiqc| 

         .. card::

            .. include:: steps_ampseq.inc

      .. tab-item:: About

         .. card::

            Amplicon sequencing (ribosomal RNA gene amplification analysis) is a highly targeted metagenomic pipeline used to analyze genetic variation in specific genomic regions. Amplicons are Polymerase Chain Reaction (PCR) products and the ultra-deep sequencing allows for efficient variant identification and characterization.

            **Uses of Amplicon sequencing**

            #. Diagnostic microbiology utilizes amplicon-based profiling that allows to sequence selected amplicons such as regions encoding 16S rRNA that are used for species identification. 

            #. Discovery of rare somatic mutations in complex samples such as tumors mixed with germline DNA.
            
            GenPipes supports the `DADA2`_ Amplicon sequencing protocol for recovering single-nucleotide resolved Amplicon Sequence Variants (ASVs) from the Amplicon data.

            See :ref:`ampschema` tab for the pipeline workflow. Check the `README.md <https://bitbucket.org/mugqic/genpipes/src/master/pipelines/ampliconseq/README.md>`_ file for implementation details.

            **References**

            * `Amplicon sequencing techniques <https://sapac.illumina.com/techniques/sequencing/dna-sequencing/targeted-resequencing/amplicon-sequencing.html>`_

            * `Amplicon Sequencing Primer <https://eurofinsgenomics.eu/en/next-generation-sequencing/amplicon-sequencing/>`_

            * `High-throughput amplicon sequencing <https://www.biorxiv.org/content/10.1101/392332v2>`_.

            * `Trimmomatic - flexible trimming <https://academic.oup.com/bioinformatics/article-pdf/30/15/2114/48924714/bioinformatics_30_15_2114.pdf>`_.

.. dropdown:: :material-outlined:`report;2em` Amplicon Sequencing Readset File
   :color: warning

   Please make sure you use the special :ref:`Amplicon Readset file format<ref_example_ampliconseq_readset_file>` and not the general readset file format.
.. The following are html links used in this text

.. _DADA2 Pipeline: https://benjjneb.github.io/dada2/tutorial.html

.. The following are replacement texts used in this file

.. |trimmomatic16S| replace:: `Trimmomatic16S Step`_
.. |merge_trimmomatic_stats16S| replace:: `Merge Trimmomatic Stats`_
.. |flash_pass1| replace:: `Flash Pass 1`_
.. |ampliconLengthParser| replace:: `Amplicon Length Parser`_
.. |flash_pass2| replace:: `Flash Pass 2`_
.. |merge_flash_stats| replace:: `Merge Flash Stats`_
.. |asva| replace:: `ASVA`_
.. |run_multiqc| replace:: `Run MultiQC`_