pique 0.1.7
An efficient peak finder for high coverage ChIP-seq experiments.
License: BSD

The pique package is a high efficiency peak finder for ChIP-seq
experiments that yield high coverage allignments to the reference
genome. It was developed for studying gene expression in Halobacterium
salinarum sp. NRC1, sequenced using barcoded 40bp Illumina reads as
part of an ongoing project in Marc Facciotti's lab at UC Davis.

AUTHOR & CONTACT INFORMATION
----------------------------

Pique was written by Russell Neches, a graduate student in Jonathan
Eisen's laboratory. 

    Russell's Blog    : http://vort.org/
    Russell's Twitter : @ryneches
    Russell's Email   : ryneches at ucdavis dot edu
    Lab homepage      : http://phylogenomics.wordpress.com/
    Project Page      : http://ryneches.github.com/pique/

INSTALLING
----------

General Instructions : You can do the usual python thing. We recommend using
virtualenv, because it's nice.

    $ cd ~
    $ virtualenv --system-site-packages opt
    $ PATH=~/opt/bin:$PATH
    $ pip install numpy         # prerequisite
    $ pip install scipy         # prerequisite
    $ pip install cython        # prerequisite
    $ pip install pysam         # prerequisite
    $ pip install argcomplete   # suggested package
    $ pip install matplotlib    # suggested package
    $ pip install ipython       # suggested package
    $ pip install pique         # install Pique
    $ pique test

Debian and Ubuntu : We recommend using apt-get for most of the
prerequisites.

    $ sudo apt-get install  python-numpy        \
                            python-scipy        \
                            python-matplotlib   \
                            python-virtualenv   \
                            cython              \
                            ipython
    $ cd ~
    $ virtualenv --system-site-packages opt
    $ PATH=~/opt/bin:$PATH
    $ pip install pysam
    $ pip install argcomplete
    $ pip install pique
    $ pique test

In your .bashrc file, we suggest adding ~/opt/bin to your PATH :
    
    $ cd ~
    $ cat >> .bashrc
    PATH=~/opt/bin:$PATH
    export PATH
    [ctrl-D]

MacOS : Unfortunately, MacOS doesn't come with virtualenv or any sort
of useful global package management system. Here's how to fix that :

    $ sudo easy_install pip     # install pip globally

Make sure pip works :

    $ pip
    Usage: pip COMMAND [OPTIONS]
    pip: error: You must give a command (use "pip help" to see a 
    list of commands)

Now, install virtualenv :

    $ sudo pip install virtualenv

Make sure that works :

    $ virtualenv
    You must provide a DEST_DIR
    ...

Now, scroll back up and follow the General Instructions.


Using argcomplete : If you installed argcomplete, you can enable tab
completion for pique commands by placing this into your .bashrc :

    eval "$(register-python-argcomplete pique)"


TESTING DATA
------------

A testing data set is [available on
Figshare](http://figshare.com/articles/A_ChIP-seq_test_dataset/98106).
It was made from a his-tagged tfbD pulldown in _Halobacterium
salinarum_, sequenced on the Illumina GAII. The reads were mapped
using [bowtie](http://bowtie-bio.sourceforge.net/) to a cut-down
chromosome and one plasmid. Unmapped reads were discarded. The dataset
can be cited separately as :

    A ChIP-seq test dataset. Russell Neches, Marc Facciotti, Elizabeth
    G. Wilbanks, Phillip M. Seitzer. **figshare**.
    Retrieved 06:12, Nov 27, 2012 (GMT)
    http://dx.doi.org/10.6084/m9.figshare.98106

If you want to try out the test data, simply run :

    $ pique test

The test data will automatically be downloaded to `/tmp/pique/`, and
the resulting output data will be written to your current working
directory.

TESTS
-----

Tests are designed to be used with nose. 

    http://somethingaboutorange.com/mrl/projects/nose/

The tests (stupidly) depend on hard-coded paths to find the test data
files, so you have to run nosetests from the main module directory
(i.e., the one that has setup.py in it).

    $ cd pique
    $ nosetests

DEPENDENCIES
------------

This package requires the following packages :

    scipy
    numpy
    pysam
    cython
        
Suggested :

    matplotlib
    ipython
    samtools
    argcomplete
    Gaggle Genome Browser

USAGE
-----

This software can be used either as a stand-alone application, or as a
library accessed by your own software.

To use pique as a stand-alone application, there is a self-documenting
command line interface :

    $ pique --help
    usage: pique [-h] {batch,tk,test,bam2wav,mapmaker} ...
    
    Pique, a ChIP-seq analytical tool.
    
    optional arguments:
      -h, --help            show this help message and exit

    commands:
      {batch,tk,test,bam2wav,mapmaker}
        batch               command-line mode for peak detection
        tk                  GUI mode for peak detection
        test                testing mode, downloads online test data
        bam2wav             BAM to WAV conversion utility
        mapmaker            genome analysis region mapping utility


The application has four modes, batch, tk, test and mapmaker. To access
the GUI interface, run :

    $ pique tk

The application window will appear. Enter a name for your project in
the 'Project name' field; this will be the name prefix for the output
files. Choose files for the IP and BG (background) tracks; these
should be indexed BAM files, created by mapping your IP and control
reads to the reference sequence. Many different tools can be used for
this, such as bowtie, bwa and SHRiMP.

The IP and background BAM files must contain identical contig names.

If desired, choose a genome map file ('Map (optional)'). See below for
how to create one.

Enter the dominant fragment length of the sequencing library, and the
read length used for the sequencing run. If you have performed quality
trimming, you may wish to use the average or median read length after
trimming for the read length.

Then, click 'Run.' Your BAM files will be loaded and processed, and
output files will be written in the directory from which pique.py was
invoked. Run time for a bacterial genome depends on the number of
mapped reads and the length of the genome. For our projects, we have
found that a run takes about one to five minutes.

BATCH MODE
----------

If you have many experiments to process, it may be more convenient to
run pique from a script. 

    $ pique batch -h
    usage: pique batch [-h] -name NAME -ip IPFILE -bg BGFILE -map MAPFILE
                       [-a ALPHA] [-l L_THRESH] [-p]

    optional arguments:
      -h, --help    show this help message and exit
      -name NAME    project name
      -ip IPFILE    BAM file for IP data
      -bg BGFILE    BAM file for control data
      -map MAPFILE  genome map file
      -a ALPHA      read length
      -l L_THRESH   expected binding footprint
      -p            make a pickel file

The arguments are essentially the same as those found in the GUI,
except for the option of making a pickle file. If this option is
enabled, after completing the anaysis, pique will dump the entire
analysis workbench object into a pickle file. This can be very useful
for debugging and data post-processing in Python. For example, the
pickle file can be used to load the analysis workbench into an
interactive ipython session and used to generate figures.

    http://docs.python.org/2/library/pickle.html

MAPMAKER MODE
-------------

    $ pique mapmaker -h
    usage: pique mapmaker [-h] -name NAME -bamfile BAMFILE [-window
    WINDOW]
                          [-stride STRIDE] -highest HIGHEST -lowest LOWEST
                          [-bins BINS]

    optional arguments:
      -h, --help        show this help message and exit
      -name NAME        project name
      -bamfile BAMFILE  BAM file of aligned data
      -window WINDOW    sliding window size
      -stride STRIDE    stride length
      -highest HIGHEST  highest histogram bin
      -lowest LOWEST    lowest histogram bin
      -bins BINS        number of histogram bins

BAM2WAV MODE
------------

As an alternative to visualization, pique contains a utility for 
converting ChIP-seq data into audio data. The background alignment 
is subtracted from the IP alignment, and a separate WAV file is 
written for each contig. Coverage information for reads that map 
in the forward and reverse orientations is represented on the left 
and right audio channels, respectively.

    $ pique bam2wav -h
    usage: pique bam2wav [-h] -name NAME -ip IPFILE -bg BGFILE
    
    optional arguments:
      -h, --help  show this help message and exit
      -name NAME  project name
      -ip IPFILE  BAM file for IP data
      -bg BGFILE  BAM file for control data


GENOME MAP FILE
---------------

The Genome Map File is a GFF-formatted file describing three kinds of
features; analysis regions, masking regions, and normalization
regions. Analysis and masking region annotations are optional;
however, each analysis region MUST have at least one normalization
region. (Note : If you do not describe any analysis regions, one will
automatically be created spanning each contig. So, if you do not
describe any analysis regions for a contig, you must still describe at
least one normalization region for that contig.)

Analysis regions should use the feature name 'analysis_region' in the
file, and describe sub-regions of the contig that you wish to process
independently. For example, if there are inconsistencies in coverage
level due to gene dosage variations with respect to the reference,
processing the dosage regions separately can improve peak detection.

Masking regions should use the feature name 'mask' in the file, and
describe sub-regions of the analysis regions that you wish to delete
from the analysis. (Note: Masking regions *must* fall completely
within an analysis region.) This is useful for hiding coverage
aberrations due to repetitive regions, such as IS elements.

By default, an analysis region describing the whole contig is created
for each contig. When an analysis region is loaded by the user via the
mapping file, the default analysis region for that contig is deleted. 

Normalization regions should use the name 'norm_region' in the map
file, and describe regions (within analysis regions) in which there
are thought to be no binding events. These regions will be used to
calibrate the sequencing coverage between the IP track and the control
track. If the user chooses poor normalization regions, Pique will
still function, but will produce incorrect normalization factors and
may set the peak detection cutoff too stringently.

Features described in the genome map file must use the same contig
names as the IP and background BAM files. To see them, run :

    $ samtools view -H BAMfile.bam

Most columns in the GFF file are unused, and may be left empty (as
long as the correct number of tabs are present). The required fields
are :

    0   contig name
    2   'analysis_region', 'mask' or 'norm_region'
    3   start coordinate
    4   end coordinate

An example genome map file is provided (data/map.gff).

OUTPUT FILES
------------

    <name>.IP.track  : A GGB-formatted track file of the IP data
    <name>.BG.track  : A GGB-formatted track file of the BG data
    <name>.bookmark  : A list of GGB bookmarks for each peak
    <name>.qp        : GGB-formatted quantitative positional data 
                       of binding coordinates
    <name>.gff       : A GFF file of each peak
    <name>.peak.tsv  : Peak statistical quantities in TSV format

The GFF output file includes the following columns :

    0   contig name
    1   software version
    2   feature description
    3   start coordinate
    4   end coordinate
    5   enrichment ratio relative to background

The TSV output includes the following columns :

    0   contig
    1   start
    2   stop
    3   binds_at            # binding coordinate
    4   enrichment_ratio    
    5   average_contig_norm # mean of all norms
    6   std_contig_norm     # standard deviation of all norms
    7..                     # all subsequent colunms are the norms

The bookmark file contains some additional annotations in addition to
the required GGB bookmark information :

    binds_at            estimated binding coordinate
    enrichment_ratio    enrichment ratio relative to background
    norm_0, norm_1...   the normalization factors calculated from the
                        normilization regions

CAVEATS
-------

Binding coordinates : The peak calling step (pique.py) will attempt to
calculate a binding coordinate for the putative peaks it finds. The
method used is extremely naive, but we find that it generally comes
within about 5bp of the expected binding site.

Double peaks : Some enriched regions contain two binding sites, but
the algorithm is unable to de-convolute them. We have tried running
these regions through several other peak callers with little success.
We have included a script for identifying sites that are likely to be
double peaks to assist with curation.

DOWNSTREAM ANALYSIS
-------------------

Pique does not attempt to filter peaks that are statistically
insignificant. We have found that this part of the analysis is rather
specific to the data and to the experiment. 

To address this, Pique is designed to achieve a low false-negative
rate at the cost of a potentially high false-positive rate (it appears
that the false-positive rate is in practice rather low compared to
other software, but we have not implemented any filtering to prevent
them from happening). The idea is that it is painstaking work to find
a putative peak, it is less difficult to discard unsatisfactory peaks
once they have been found.  Thus, some kind of post-filtering is
necessary. Pique provides the user with output that can be used to
support a variety of such statistical tests.

Some recommended filtering might include :

    [1] Eliminate peaks that are significantly narrower than the size
        range of the sequencing library, and perhaps other peaks that
        cluster with them.

    [2] Eliminate peaks with a normalized enrichment ratio below 1.0,
        or peaks that cluster with them.

    [3] Eliminate peaks that have predicted binding sites that are
        very skewed from the center of the enriched region.

Depending on how many peaks are recovered, the user may wish to try
one or all of these, perhaps with clustering to avoid arbitrary
cutoffs.

However, we do not recommend trying to generate a "perfect" peak list
by statistical analysis alone.

MANUAL CURATION
---------------

Microbial genomes are fairly small. It is probably not worth the
effort to undertake a very complicated statistical effort to eliminate
all false positives. We recommend applying a few basic statistical
tests, and then loading tracks, quantitative positionals and bookmarks
into the Gaggle Genome Browser.

Once the data is loaded into the browser, view the bookmark list. When
you select a bookmark, the browser will automatically scan to that
region of the genome and highlight the putative peak region. If you
think the peak is a false positive, press the delete key.

When you are finished, save a copy of the bookmark file. We find that
curation of a microbial genome in this way takes about ten to twenty
minutes.

INSPIRATIONAL QUOTE
-------------------

I met men at every turn who owned from one thousand to thirty thousand
"feet" in undeveloped silver mines, every single foot of which they
believed would shortly be worth from fifty to a thousand dollars—and
as often as any other way they were men who had not twenty-five
dollars in the world. Every man you met had his new mine to boast of,
and his "specimens" ready; and if the opportunity offered, he would
infallibly back you into a corner and offer as a favor to you, not to
him, to part with just a few feet in the "Golden Age," or the "Sarah
Jane," or some other unknown stack of croppings, for money enough to
get a "square meal" with, as the phrase went. And you were never to
reveal that he had made you the offer at such a ruinous price, for it
was only out of friendship for you that he was willing to make the
sacrifice. Then he would fish a piece of rock out of his pocket, and
after looking mysteriously around as if he feared he might be waylaid
and robbed if caught with such wealth in his possession, he would dab
the rock against his tongue, clap an eyeglass to it, and exclaim:

"Look at that! Right there in that red dirt! See it? See the specks of
gold? And the streak of silver? That's from the Uncle Abe. There's a
hundred thousand tons like that in sight! Right in sight, mind you!
And when we get down on it and the ledge comes in solid, it will be
the richest thing in the world! Look at the assay! I don't want you to
believe me—look at the assay!"

Then he would get out a greasy sheet of paper which showed that the
portion of rock assayed had given evidence of containing silver and
gold in the proportion of so many hundreds or thousands of dollars to
the ton.

I little knew, then, that the custom was to hunt out the richest piece
of rock and get it assayed! Very often, that piece, the size of a
filbert, was the only fragment in a ton that had a particle of metal
in it—and yet the assay made it pretend to represent the average value
of the ton of rubbish it came from!

    - Mark Twain, Roughing It


LEGAL STUFF
-----------

Copyright (c) 2012, Russell Neches
All rights reserved.

Redistribution and use in source and binary forms, with or without
modification, are permitted provided that the following conditions are
met:

    * Redistributions of source code must retain the above copyright 
      notice, this list of conditions and the following disclaimer.

    * Redistributions in binary form must reproduce the above copyright
      notice, this list of conditions and the following disclaimer in 
      the documentation and/or other materials provided with the 
      distribution.

    * Neither the name of the University of California, Davis nor the 
      names of its contributors may be used to endorse or promote 
      products derived from this software without specific prior 
      written permission.

THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS
"AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT
LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR
A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT
HOLDER OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL,
SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT
LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE,
DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY
THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT
(INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE
OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.