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|
.. _basic:
Basic analysis
--------------
.. _CalcBasicStats:
CalcBasicStats
^^^^^^^^^^^^^^
This routine computes a set of basic sample statistics, such as read
counts, number of clonotypes, etc.
Command line usage
~~~~~~~~~~~~~~~~~~
.. code-block:: bash
$VDJTOOLS CalcBasicStats \
[options] [sample1.txt sample2.txt ... if -m is not specified] output_prefix
Parameters:
+-------------+-----------------------+------------+--------------------------------------------------------------------+
| Shorthand | Long name | Argument | Description |
+=============+=======================+============+====================================================================+
| ``-m`` | ``--metadata`` | path | Path to metadata file. See :ref:`common_params` |
+-------------+-----------------------+------------+--------------------------------------------------------------------+
| ``-u`` | ``--unweighted`` | | If not set, all statistics will be weighted by clonotype frequency |
+-------------+-----------------------+------------+--------------------------------------------------------------------+
| ``-h`` | ``--help`` | | Display help message |
+-------------+-----------------------+------------+--------------------------------------------------------------------+
Tabular output
~~~~~~~~~~~~~~
The following table with ``.basicstats.txt`` suffix is generated,
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| Column | Description |
+========================+====================================================================================================================================================================+
| sample\_id | Sample unique identifier |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| ... | Metadata columns. See :ref:`metadata` section |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| count | Number of reads in a given sample |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| diversity | Number of clonotypes in a given sample |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| mean\_frequency | Mean clonotype frequency |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| geomean\_frequency | Geometric mean of clonotype frequency |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| nc\_diversity | Number of non-coding clonotypes |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| nc\_frequency | Frequency of reads that belong to non-coding clonotypes |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| mean\_cdr3nt\_length | Mean length of CDR3 nucleotide sequence. Weighted by clonotype frequency |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| mean\_insert\_size | Mean number of inserted random nucleotides in CDR3 sequence. Characterizes V-J insert for receptor chains without D segment, or a sum of V-D and D-J insert sizes |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| mean\_ndn\_size | Mean number of nucleotides that lie between V and J segment sequences in CDR3 |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
| convergence | Mean number of unique CDR3 nucleotide sequences that code for the same CDR3 amino acid sequence |
+------------------------+--------------------------------------------------------------------------------------------------------------------------------------------------------------------+
Graphical output
~~~~~~~~~~~~~~~~
none
--------------
.. _CalcSegmentUsage:
CalcSegmentUsage
^^^^^^^^^^^^^^^^
This routine computes Variable (V) and Joining (J) segment usage
vectors, i.e. the frequency of associated reads for each of V/J segments
present in sample(s). If plotting is on, will also perform clustering
for V/J usage vectors and samples *à la* gene expression analysis.
Command line usage
~~~~~~~~~~~~~~~~~~
.. code-block:: bash
$VDJTOOLS CalcSegmentUsage \
[options] [sample1.txt sample2.txt ... if -m is not specified] output_prefix
Parameters:
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------+
| Shorthand | Long name | Argument | Description |
+=============+=======================+============+=============================================================================================================+
| ``-m`` | ``--metadata`` | path | Path to metadata file. See :ref:`common_params` |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------+
| ``-u`` | ``--unweighted`` | | Will compute the number of unique clonotypes with a given V/J segment. Counts the number of reads otherwise |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------+
| ``-p`` | ``--plot`` | | Turns on plotting. See :ref:`common_params` |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------+
| ``-f`` | ``--factor`` | string | Specifies plotting factor. See :ref:`common_params` |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------+
| ``-n`` | ``--numeric`` | | Specifies if plotting factor is numeric. See :ref:`common_params` |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------+
| ``-l`` | ``--label`` | string | Specifies label used for plotting. See :ref:`common_params` |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------+
| ``-h`` | ``--help`` | | Display help message |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------+
Tabular output
~~~~~~~~~~~~~~
The following tables with
``.segments.[unwt or wt depending on -u parameter].[V or J].txt`` suffix
are generated,
+-----------------------------------+------------------------------------------------+
| Column | Description |
+===================================+================================================+
| sample\_id | Sample unique identifier |
+-----------------------------------+------------------------------------------------+
| ... | Metadata columns. See :ref:`metadata` section |
+-----------------------------------+------------------------------------------------+
| Segment name, e.g. TRBJ1-1 | Segment frequency in a given sample |
+-----------------------------------+------------------------------------------------+
| Next segment name, e.g. TRBJ1-2 | ... |
+-----------------------------------+------------------------------------------------+
| ... | ... |
+-----------------------------------+------------------------------------------------+
Graphical output
~~~~~~~~~~~~~~~~
Images, having the same name as tables, with the exception of ``.pdf``
extension, are created if plotting is on. They display segment usage
heatmap and hierarchical clustering for samples and segment.
This figure will be created using `heatmap.2` function from
`gplots <http://cran.r-project.org/web/packages/gplots/>`__ R package
with default clustering parameters.
.. figure:: _static/images/modules/basic-segmentusage.png
:align: center
:scale: 50 %
**Sample clustering based on Variable segment usage**. Weighted
Variable usage profiles are used, hierarchical clustering is
performed using euclidean distance. A continuous factor is displayed
(``-n -f age`` argument).
--------------
.. _CalcSpectratype:
CalcSpectratype
^^^^^^^^^^^^^^^
Calculates
`spectratype <http://www.jimmunol.org/content/152/10/5109.full.pdf+html>`__,
that is, histogram of read counts by CDR3 nucleotide length. The
spectratype is useful to detect pathological and highly clonal
repertoires, as the spectratype of non-expanded T- and B-cells has a
symmetric gaussian-like distribution.
Command line usage
~~~~~~~~~~~~~~~~~~
.. code-block:: bash
$VDJTOOLS CalcSpectratype \
[options] [sample1.txt sample2.txt ... if -m is not specified] output_prefix
Parameters:
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------------------+
| Shorthand | Long name | Argument | Description |
+=============+=======================+============+=========================================================================================================================+
| ``-m`` | ``--metadata`` | path | Path to metadata file. See :ref:`common_params` |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------------------+
| ``-u`` | ``--unweighted`` | | Instead of computing read frequency, will compute the number of unique clonotypes with specific a CDR3 length |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------------------+
| ``-a`` | ``--amino-acid`` | | Will use CDR3 amino acid sequences for calculation instead of nucleotide ones |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------------------+
| ``-h`` | ``--help`` | | Display help message |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------------------------------------+
Tabular output
~~~~~~~~~~~~~~
The following table with
``.spectratype.[aa or nt depending on -a parameter].[unwt or wt depending on -u parameter].txt``
suffix is generated,
+------------------------+------------------------------------------------------------------+
| Column | Description |
+========================+==================================================================+
| sample\_id | Sample unique identifier |
+------------------------+------------------------------------------------------------------+
| ... | Metadata columns. See :ref:`metadata` section |
+------------------------+------------------------------------------------------------------+
| CDR3 length, e.g. 22 | Frequency of reads with a given CDR3 length in a given sample |
+------------------------+------------------------------------------------------------------+
| Next CDR3 length, 23 | ... |
+------------------------+------------------------------------------------------------------+
| ... | ... |
+------------------------+------------------------------------------------------------------+
Graphical output
~~~~~~~~~~~~~~~~
none
--------------
.. _PlotFancySpectratype:
PlotFancySpectratype
^^^^^^^^^^^^^^^^^^^^
Plots a spectratype that also displays CDR3 lengths for top N clonotypes
in a given sample. This plot allows to detect the highly-expanded
clonotypes.
Command line usage
~~~~~~~~~~~~~~~~~~
.. code-block:: bash
$VDJTOOLS PlotFancySpectratype [options] sample.txt output_prefix
Parameters:
+-------------+-----------------------+------------+----------------------------------------------------------------------------+
| Shorthand | Long name | Argument | Description |
+=============+=======================+============+============================================================================+
| ``-t`` | ``--top`` | int | Number of top clonotypes to visualize. Should not exceed 20, default is 10 |
+-------------+-----------------------+------------+----------------------------------------------------------------------------+
| ``-h`` | ``--help`` | | Display help message |
+-------------+-----------------------+------------+----------------------------------------------------------------------------+
Tabular output
~~~~~~~~~~~~~~
Following table with ``.fancyspectra.txt`` prefix is generated,
+-------------------------------------+----------------------------------------------------------------------+
| Column | Description |
+=====================================+======================================================================+
| Len | Length of CDR3 nucleotide sequence |
+-------------------------------------+----------------------------------------------------------------------+
| Other | Frequency of clonotypes with a given CDR3 length, other than top N |
+-------------------------------------+----------------------------------------------------------------------+
| Clonotype#N, e.g. CASRLLRAGSTEAFF | Clonotype frequency, at the corresponding CDR3 length |
+-------------------------------------+----------------------------------------------------------------------+
| Clonotype#N-1 | ... |
+-------------------------------------+----------------------------------------------------------------------+
| ... | ... |
+-------------------------------------+----------------------------------------------------------------------+
Graphical output
~~~~~~~~~~~~~~~~
The following image file with ``.fancyspectra.pdf`` suffix,
.. figure:: _static/images/modules/basic-fancyspectra.png
:align: center
:scale: 50 %
**Spectratype with additional detalization.**
Most abundant clonotypes are explicitly shown.
--------------
.. _PlotFancyVJUsage:
PlotFancyVJUsage
^^^^^^^^^^^^^^^^
Plots a `circos <http://circos.ca/>`__-style V-J usage plot displaying
the frequency of various V-J junctions.
Command line usage
~~~~~~~~~~~~~~~~~~
.. code-block:: bash
$VDJTOOLS PlotFancyVJUsage [options] sample.txt output_prefix
Parameters:
+-------------+-----------------------+------------+-----------------------------------------------------------------------------------------------------------------+
| Shorthand | Long name | Argument | Description |
+=============+=======================+============+=================================================================================================================+
| ``-u`` | ``--unweighted`` | | Instead of computing read frequency, will compute the number of unique clonotypes with specific V-J junctions |
+-------------+-----------------------+------------+-----------------------------------------------------------------------------------------------------------------+
| ``-h`` | ``--help`` | | Display help message |
+-------------+-----------------------+------------+-----------------------------------------------------------------------------------------------------------------+
Tabular output
~~~~~~~~~~~~~~
A matrix with rows corresponding to different J segments and columns
corresponding to different V segments. Each cells contains the frequency
of a give V-J junction. The file has
``.fancyvj.[unwt or wt depending on -u parameter].txt`` suffix.
Graphical output
~~~~~~~~~~~~~~~~
An image having the same name as the output table, with the exception of
``.pdf`` extension, is generated. The plot is built using
`circlize <http://cran.r-project.org/web/packages/circlize/>`__ R package.
.. figure:: _static/images/modules/basic-fancyvj.png
:align: center
:scale: 50 %
**V-J junction circos plot for a single sample.**
Arcs correspond to different V and J segments, scaled to their frequency
in sample. Ribbons represent V-J pairings and their size is scaled to
the pairing frequency (weighted in present case).
--------------
.. _PlotSpectratypeV:
PlotSpectratypeV
^^^^^^^^^^^^^^^^
Plots a detailed spectratype containing additional info displays CDR3
length distribution for clonotypes from top N Variable segment families.
This plot is useful to detect type 1 and type 2 repertoire
`biases <http://www.nature.com/nri/journal/v6/n12/fig_tab/nri1977_T1.html>`__,
that could arise under pathological conditions.
Command line usage
~~~~~~~~~~~~~~~~~~
.. code-block:: bash
$VDJTOOLS PlotSpectratypeV [options] sample.txt output_prefix
Parameters
~~~~~~~~~~
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------+
| Shorthand | Long name | Argument | Description |
+=============+=======================+============+===========================================================================================+
| ``-t`` | ``--top`` | int | Number of top (by frequency) V segments to visualize. Should not exceed 12 default is 12 |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------+
| ``-u`` | ``--unweighted`` | | Instead of counting read frequency, will count the number of unique clonotypes |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------+
| ``-h`` | ``--help`` | | Display help message |
+-------------+-----------------------+------------+-------------------------------------------------------------------------------------------+
**Tabular output**
Following table with
``.spectraV.[unwt or wt depending on -u parameter].txt`` prefix is
generated,
+----------------------------+--------------------------------------------------------------------------------------------+
| Column | Description |
+============================+============================================================================================+
| Len | Length of CDR3 nucleotide sequence |
+----------------------------+--------------------------------------------------------------------------------------------+
| Other | Frequency of clonotypes with a given CDR3 length, having V segments other than the top N |
+----------------------------+--------------------------------------------------------------------------------------------+
| Segment#N, e.g. TRBV10-1 | Frequency of clonotypes with a given V segment at the corresponding CDR3 length |
+----------------------------+--------------------------------------------------------------------------------------------+
| Segment#N-1 | ... |
+----------------------------+--------------------------------------------------------------------------------------------+
| ... | ... |
+----------------------------+--------------------------------------------------------------------------------------------+
**Graphical output**
The following image file with
``.spectraV.[unwt or wt depending on -u parameter].pdf`` suffix,
.. figure:: _static/images/modules/basic-spectrav.png
:align: center
:scale: 50 %
**Stacked spectratypes by Variable segment for a single sample.**
Most frequent Variable segments are highlighted.
|
