<tool id="LEfSe_for" name="A) Format Data for LEfSe" version="1.0">
  <code file="format_input_selector.py"/> 
	<description></description>
<!-- <command interpreter="python">./format_input.py $inp_data $formatted_input -f $feat_dir -c $cls_n -s $subcls_n -u $subj_n -o 1000000.0 </command>   -->
<command interpreter="python">format_input.py $inp_data $formatted_input -f $cond.feat_dir -c $cond.cls_n -s $cond.subcls_n -u $cond.subj_n -o $norm </command> 
  <inputs>
    <page>
	<param format="tabular" name="inp_data" type="data" label="Upload a tabular file of relative abundances and class labels (possibly also subclass and subjects labels) for LEfSe - See samples below - Please use Galaxy Get-Data/Upload-File. Use File-Type = Tabular" help=""/>
	<param name="cond" type="data_column" data_ref="inp_data" accept_default="true" /> 

	<conditional name="cond" type="data_column" data_ref="inp_data" accept_default="true">
        <param name="feat_dir" type="select" data_ref="inp_data" label="Select whether the vectors (features and meta-data information) are listed in rows or columns" help="">
          <option value="r" selected='True'>Rows</option>
          <option value="c">Columns</option>
        </param>

        <when value="r">
                <param name="cls_n" label="Select which row to use as class" size ="70" type='select' dynamic_options="get_cols(inp_data,'r','cl')" />
                <param name="subcls_n" label="Select which row to use as subclass" type='select' dynamic_options="get_cols(inp_data,'r','subclass')" />
                <param name="subj_n" label="Select which row to use as subject" type='select' dynamic_options="get_cols(inp_data,'r','subject')" />
        </when>
        <when value="c">
                <param name="cls_n" label="Select which column to use as class" type='select' dynamic_options="get_cols(inp_data,'c','cl')" />
                <param name="subcls_n" label="Select which column to use as subclass" type='select' dynamic_options="get_cols(inp_data,'c','subclass')" />
                <param name="subj_n" label="Select which column to use as subject" type='select' dynamic_options="get_cols(inp_data,'c','subject')" />
        </when>

        </conditional>

	<param name="norm" type="select" label="Per-sample normalization of the sum of the values to 1M (recommended when very low values are present)" help="">
          <option value="1000000.0" selected='True'>Yes</option>
          <option value="-1">No</option>
        </param>

<!--	<param name="row" label="on row" type="data_row" data_ref="inp_data" accept_default="true" /> -->
    </page>
   </inputs>
  <outputs>
    <data format="lefse" name="formatted_input" />
  </outputs>
  
  <tests>
    <test>
      <param name="inp_data" value="lefse_input"  ftype="tabular"  />
      <param name="cond.feat_dir" value="r" />
	  <param name="cond.cls_n" value="1"  />
	  <param name="cond.subcls" value="-1"  />
	  <param name="cond.subj" value="-1"  />
	  <param name="norm" value="1000000"  />
      <output name="formatted_input" file="lefse_output_a"  />
    </test>
  </tests>   
 
  
  
  
<help>
	
	
**What it does**

LDA Effect Size (LEfSe) `(Segata et. al 2010)`_ is an algorithm for high-dimensional biomarker discovery and 
explanation that identifies genomic features (genes, pathways, or taxa) characterizing 
the differences between two or more biological conditions (or classes, see figure below). It 
emphasizes both statistical significance and biological relevance, allowing 
researchers to identify differentially abundant features that are also consistent with 
biologically meaningful categories (subclasses). LEfSe first robustly 
identifies features that are statistically different among biological classes. It then 
performs additional tests to assess whether these differences are consistent with 
respect to expected biological behavior. 

Specifically, we first use the non-parametric factorial 
Kruskal-Wallis (KW) sum-rank test to detect features with 
significant differential abundance with respect to the class of interest; biological 
significance is subsequently investigated using a set of pairwise tests among 
subclasses using the (unpaired) Wilcoxon rank-sum test. As a last step, LEfSe uses 
Linear Discriminant Analysis to estimate the effect size of each differentially 
abundant feature and, if desired by the investigator, to perform dimension reduction.

LEfSe consists of six modules performing the following steps (see the figure below).

The first step consists of **uploading your file** by using Galaxy's "Get-Data / Upload-file"


The next steps are:

+ **A) Format Data for LEfSe**: selects the structure of the problem (classes, subclasses, subjects) and formats the tabular abundance data for the B module
+ **B) LDA Effect Size (LEfSe)**: performs the analysis using the data formatted with module A and provides input for the visualization modules (C, D, E, F)
+ **C) Plot LEfSe Results**: graphically reports the discovered biomarkes (output of B) with their effect sizes
+ **D) Plot Cladogram**: graphically represents the discovered biomarkers (output of B) in a taxonomic tree specified by the hierarchical feature names (not available for non-hierarchical features)
+ **E) Plot One Feature**: plots the row values of a feature (biomarker or not) as an abundance histogram with classes and subclasses structure (only one feature at the time)
+ **F) Plot Differential Features**: plots the row values of all features (biomarkers or not) as abundance histograms with classes and subclasses structure and provides a zip archive of the figures

.. image:: https://bytebucket.org/biobakery/galaxy_lefse/wiki/lefse_ove.png


------


**Input file format**

The text tab-delimited input file consists of a list of numerical features, the class vector and optionally the subclass and subject vectors. The features can be read counts directly or abundance floating-point values more generally, and the first field is the name of the feature. Class, subclass and subject vectors have a name (the first field) and a list of non-numerical strings.

Although both column and row feature organization is accepted, given the high-dimensional nature of metagenomic data, the listing of the features in rows is preferred. A partial example of an input file follows (all values are separated by single-tab)::

	bodysite				mucosal		mucosal		mucosal		mucosal		mucosal		non_mucosal	non_mucosal	non_mucosal	non_mucosal	non_mucosal
	subsite					oral		gut		oral		oral		gut		skin		nasal		skin		ear		nasal
	id					1023		1023		1672		1876		1672		159005010	1023		1023		1023		1672
	Bacteria				0.99999		0.99999		0.999993	0.999989	0.999997	0.999927	0.999977	0.999987	0.999997	0.999993
	Bacteria|Actinobacteria			0.311037	0.000864363	0.00446132	0.0312045	0.000773642	0.359354	0.761108	0.603002	0.95913		0.753688
	Bacteria|Bacteroidetes			0.0689602	0.804293	0.00983343	0.0303561	0.859838	0.0195298	0.0212741	0.145729	0.0115617	0.0114511
	Bacteria|Firmicutes			0.494223	0.173411	0.715345	0.813046	0.124552	0.177961	0.189178	0.188964	0.0226835	0.192665
	Bacteria|Proteobacteria			0.0914284	0.0180378	0.265664	0.109549	0.00941215	0.430869	0.0225884	0.0532684	0.00512034	0.0365453
	Bacteria|Firmicutes|Clostridia		0.090041	0.170246	0.00483188	0.0465328	0.122702	0.0402301	0.0460614	0.135201	0.0115835	0.0537381

In this case one may want to use bodysite as class, subsite as subclass and id as subject. Notice that the features have a hierarchical structure specified using the character \|.


**Input file sample**

You can try the LEfSe modules using the dataset available here_.   You can upload the dataset using Galaxy's  **Get-Data / Upload File**

This is a 16S dataset from `(Garrett et. al 2010)`_ and `(Veiga et. al 2010)`_ for studying the characteristics of the fecal microbiota in a mouse model of spontaneous colitis. The dataset contains 30 abundance profiles (obtained processing the 16S reads with RDP) belonging to 10 rag2 (control) and 20 truc (case) mice. The metadata consists in class information only, as we don't have subject or subclass information. The same dataset is used to show the graphical results in the module descriptions.



------

STEP A:
-------	
	
	
**What STEP A does**

Preprocessing module for the biomarker discovery tool called LEfSe:

This module of LEfSe preprocesses metagenomic abundance data for the analyses to be carried out with the "Run LEfSe" module. This module is separated from the "Run LEfSe" because one may want to preprocess the data only once but run multiple analyses.

For an overview of LEfSe please refer to the "Introduction" module or to `(Segata et. al 2011)`_.

------

**Input format**

The module accepts tabular data with the feature list in rows or columns.

------

**Output format**

The module generates data readable by the "Run LEfSe" module only. 

------

**Parameters**

The class vector represents the labels of the main condition under investigation. The (optional) subclass vector denotes the internal groupings with biological meaning inside each class (subclasses of different classes with the same name are processed as different subclasses). The subject vector (optional) reports a third dimension denoting meta-data (subject id, sample type, ... ) which is independent from the class and subclass definition. 

The labels can have a hierarchical organization (see example below) reflecting taxonomies (like NCBI or RDB microbial taxonomy, SEED subsystems or GO terms). The taxonomic levels are specified using the character \|.

The per-sample normalization is usually applied for metagenomic data in which the relative abundances are taken into account. 

------

**Example**

Although both column and row feature organization is accepted, given the high-dimensional nature of metagenomic data, the listing of the features in rows is preferred. A partial example of an input file follows (all values are separated by single-tab)::

	bodysite				mucosal		mucosal		mucosal		mucosal		mucosal		non_mucosal	non_mucosal	non_mucosal	non_mucosal	non_mucosal
	subsite					oral		gut		oral		oral		gut		skin		nasal		skin		ear		nasal
	id					1023		1023		1672		1876		1672		159005010	1023		1023		1023		1672
	Bacteria				0.99999		0.99999		0.999993	0.999989	0.999997	0.999927	0.999977	0.999987	0.999997	0.999993
	Bacteria|Actinobacteria			0.311037	0.000864363	0.00446132	0.0312045	0.000773642	0.359354	0.761108	0.603002	0.95913		0.753688
	Bacteria|Bacteroidetes			0.0689602	0.804293	0.00983343	0.0303561	0.859838	0.0195298	0.0212741	0.145729	0.0115617	0.0114511
	Bacteria|Firmicutes			0.494223	0.173411	0.715345	0.813046	0.124552	0.177961	0.189178	0.188964	0.0226835	0.192665
	Bacteria|Proteobacteria			0.0914284	0.0180378	0.265664	0.109549	0.00941215	0.430869	0.0225884	0.0532684	0.00512034	0.0365453
	Bacteria|Firmicutes|Clostridia		0.090041	0.170246	0.00483188	0.0465328	0.122702	0.0402301	0.0460614	0.135201	0.0115835	0.0537381

In this case one may want to use bodysite as class, subsite as subclass and id as subject. Notice that the features have a hierarchical structure specified using the character \|.

**Example with the "mouse model dataset"**

You can try the LEfSe modules using the dataset available here_. This is a 16S dataset from `(Garrett et. al 2010)`_ and `(Veiga et. al 2010)`_ for studying the characteristics of the fecal microbiota in a mouse model of spontaneous colitis. The dataset contains 30 abundance profiles (obtained processing the 16S reads with RDP) belonging to 10 rag2 (control) and 20 truc (case) mice. The metadata consists of class information only, as we don't have subject or subclass information. The dataset contains the features organized in rows; you need to select the first row as class, whereas you have to select "no subclass" and "no subject" options.
        
    
.. _here: http://www.huttenhower.org/webfm_send/73
.. _(Segata et. al 2011): http://www.ncbi.nlm.nih.gov/pubmed/21702898
.. _(Garrett et. al 2010): http://www.ncbi.nlm.nih.gov/pubmed/20833380
.. _(Veiga et. al 2010): http://www.ncbi.nlm.nih.gov/pubmed/20921388
.. _contact us: nsegata@hsph.harvard.edu




**How to Cite LEfSe**

If you find LEfSe usefull in your research please city our paper `(Segata et. al 2010)`_:

| `Nicola Segata`_, Jacques Izard, Levi Walron, Dirk Gevers, Larisa Miropolsky, Wendy Garrett, `Curtis Huttenhower`_.
| "`Metagenomic Biomarker Discovery and Explanation`_" 
| Genome Biology, 2011 Jun 24;12(6):R60


Please do not hesitate to `contact us`_ for any questions of comments. 

.. _here: http://www.huttenhower.org/webfm_send/73
.. _(Segata et. al 2010): http://www.ncbi.nlm.nih.gov/pubmed/21702898
.. _(Garrett et. al 2010): http://www.ncbi.nlm.nih.gov/pubmed/20833380
.. _(Veiga et. al 2010): http://www.ncbi.nlm.nih.gov/pubmed/20921388
.. _contact us: nsegata@hsph.harvard.edu
.. _Nicola Segata: nsegata@hsph.harvard.edu
.. _Curtis Huttenhower: chuttenh@hsph.harvard.edu
.. _Metagenomic Biomarker Discovery and Explanation: http://genomebiology.com/2011/12/6/R60 	
	
	
	

</help>
</tool>