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<head><meta http-equiv="Content-Type" content="text/html; charset=ISO-8859-1">
<title>AntEvolo 0.1 manual</title>
<style type="text/css">
.code {
background-color: lightgray;
}
</style>
<style>
</style>
</head>
<h1>AntEvolo v0.1 manual</h1>
1. <a href="#intro">About AntEvolo</a><br>
1.1 <a href = "#about_antevolo">About AntEvolo</a><br>
1.2 <a href = "#about_tree_visualizer">About TreeVisualizer</a><br>
2. <a href="#install">Installation</a><br>
2.1. <a href="#test_datasets">Verifying your installation</a><br>
3. <a href="#antevolo_usage">AntEvolo usage</a><br>
3.1. <a href="#antevolo_basic">Basic options</a><br>
3.2. <a href="#antevolo_advanced">Advanced options</a><br>
3.3. <a href="#antevolo_examples">Examples</a><br>
3.4. <a href="#antevolo_output">Output files</a><br>
4. <a href = "#treevisualizer_usage">TreeVisualizer usage</a><br>
4.1. <a href = "#visualizer_basic">Basic options</a><br>
4.2. <a href = "#visualizer_examples">Examples</a><br>
4.3. <a href = "#visualizer_output">Output files</a><br>
5. <a href = "#examples">Examples</a><br>
6. <a href="#output_files">Antibody repertoire representation</a><br>
6.1. <a href="#clusters_fasta">CLUSTERS.FASTA file format</a><br>
6.2. <a href="#read_cluster_map">RCM file format</a><br>
6.3. <a href="#alignment_info">Alignment Info file format</a><br>
7. <a href="#feedback">Feedback and bug reports</a><br>
7.1. <a href="#citation">Citation</a><br>
<!--- ----------------- -->
<a id="intro"></a>
<h2>1. About AntEvolo</h2>
AntEvolo is a tool for reconstruction of B-cell clonal lineages. The pipeline consists of two parts:
<ul>
<li><b>AntEvolo</b> itself.</li>
<li><b>TreeVisualizer</b> — a tool for drawing clonal trees images using <b>dot</b>.</li>
</ul>
<!--- ---------------------------------------------------------------- -->
<h3 id = "about_antevolo">About AntEvolo</h3>
IgReC pipeline in shown below:<br>
<p align = center>
<img src="docs/manual_figs/igrec.png" alt="igrec_pipeline" width = 70%>
</p>
<h4>Input:</h4>
<p align="justify">
AntEvolo takes as an input corrected Illumina reads.
<b>Please note that AntEvolo constructs expects that input reads cover variable region of antibody.</b>
</p>
<h4>Output:</h4>
<p align = justify>
AntEvolo partitions reads into clusters corresponding to clonal lineages and in each cluster reconstructs clonal forest.
AntEvolo provides user with the following information about constructed forests:
</p>
<ul>
<li>Clonal lineage clusters:</li>
<ul>
<li><i>clonal_trees</i> dir contains tsv dataframes with informaton about in-cluster edges,</li>
<li><i>clonal_trees_vertices</i> dir contains tsv dataframes with informaton about cluster's vertices;</li>
</ul>
Please note that tsv files are named in the following format: <b>clonal_tree_VJCLASSINDEX_CONNECTEDCOMPONENTINDEX_TREEINDEX.tree</b>
<li>CDRs information: a tsv dataframe cdr_details.txt</li>
<li>SHMs information: shm_details.txt</li>
<li>Some trees statistics: a tsv dataframe tree_details.txt</li>
</ul>
<h4>Stages:</h4>
AntEvolo pipeline consists of the following steps:
<ol>
<li><b>VJ Finder</b>: cleaning input reads using alignment against Ig germline genes</li>
<li><b>IG Trie Compressorr</b>: collects unique prefixes of the cleaned reads</li>
<li><b>Clonal trees construction</b> </li>
</ol>
<!--- ---------------------------------------------------------------- -->
<h3 id = "about_tree_visualizer">About TreeVisualizer</h3>
<h4>Input:</h4>
<p align="justify">
TreeVisualizer takes as an input a directory with AntEvolo results (or any directory containing consistent clonal_trees and clonal_trees_vertices subdirectories).
</p>
<h4>Output:</h4>
<p align = justify>
For each tree, TreeVisualizer creates a file with graph information in <b>dot</b> format and a pdf image.
</p>
<!--- ---------------------------------------------------------------- -->
<!--- ---------------------------------------------------------------- -->
<a id="install"></a>
<h2>2. Installation</h2>
AntEvolo has the following dependencies:
<ul>
<li>64-bit Linux system</li>
<li>g++ (version 4.7 or higher)</li>
<li>cmake (version 2.8.8 or higher)</li>
<li>Python 2 (version 2.7 or higher), including:</li>
<ul>
<li><a href = "http://biopython.org/wiki/Download">BioPython</a></li>
<li><a href = "http://matplotlib.org/users/installing.html">Matplotlib</a></li>
<li><a href = "http://www.numpy.org/">NumPy</a></li>
<li><a href = "http://www.scipy.org/install.html">SciPy</a></li>
</ul>
</ul>
TreeVisualizer also has a dependence on
<ul>
<li><a href = "http://www.graphviz.org/Download..php">Graphviz</a>'s dot (the command-line tool, not Python package) </li>
</ul>
To install AntEvolo, type:
<pre class="code">
<code>
./prepare_cfg
</code>
</pre>
and:
<pre class="code">
<code>
make ant
</code>
</pre>
<a id="test_datasets"></a>
<h3>2.1. Verifying your installation</h3>
For testing purposes, AntEvolo comes with toy data set. <br><br>
► To try AntEvolo on the test data set, run:
<pre class="code"><code>
./antevolo.py --test
</code>
</pre>
If the installation of AntEvolo is successful, you will find the following information at the end of the log:
<pre class="code">
<code>
Thank you for using AntEvolo!
Log was written to installation_directory/antevolo_test/antevolo.log
</code>
</pre>
<!--- ---------------------------------------------------------------- -->
<!--- ---------------------------------------------------------------- -->
<a id="antevolo_usage"></a>
<h2>3. AntEvolo usage</h2>
<p>
AntEvolo takes as an input Illumina reads covering variable region of antibody, splits them into clonal lineages and reconstructs child-parent relations.
</p>
To run AntEvolo, type:
<pre class="code">
<code>
./antevolo.py [options] -i <input_reads.fastq> -o <output_dir>
</code>
</pre>
<!--- --------------------- -->
<a id="antevolo_basic"></a>
<h3>3.1. Basic options:</h3>
<code>-i <input_reads.fastq></code><br>
FASTQ file with single Illumina reads (required).
<br><br>
<code>-o / --output <output_dir></code><br>
Output directory (required).
<br><br>
<code>-t / --threads <int></code><br>
The number of parallel threads. The default value is <code>16</code>.
<br><br>
<code>--test</code><br>
Running on the toy test dataset. Command line corresponding to the test run is equivalent to the following:
<pre class = "code">
<code>
./antevolo.py -i test_dataset/merged_reads.fastq -l IG -o antevolo_test
</code>
</pre>
<code>--help</code><br>
Printing help.
<br><br>
<!--- --------------------- -->
<a id="antevolo_advanced"></a>
<h3>3.2. Advanced options:</h3>
<code>--loci / -l <str></code><br>
Immunological loci to align input reads and discard reads with low score (required). <br>
Available values are <code>IGH</code> / <code>IGL</code> / <code>IGK</code> / <code>IG</code> (for all BCRs)</code>.
<br><br>
<code>--tau <int></code><br>
Maximum allowed number of mismatches between two reads corresponding to identical antibodies. The default (and recommended) value is 3.
Reasonable value of <code>tau</code> lies between <code>1</code> and <code>6</code>.
<br>
<b>This option is temporarily disabled!</b>
<br><br>
<!--- --------------------- -->
<a id="antevolo_examples"></a>
<h3>3.3. Examples</h3>
To reconconstruct clonal trees from single reads <code>reads.fastq</code>, type:
<pre class="code">
<code>
./antevolo.py -i reads.fastq -o output_dir
</code>
</pre>
<!--- --------------------- -->
<a id="antevolo_output"></a>
<h3>3.4. Output files</h3>
AntEvolo creates working directory (which name was specified using option <code>-o</code>)
and outputs the following files there:
<ul>
<li>Subdirectories with information about trees:</li>
<ul>
<li><b>clonal_trees/</b>
<ul>
<li> clonal_trees/clonal_tree_1-14-2.tree — a tsv dataframe for each cluster/tree containing all its edges (details in <a href= "#output_files">Clonal trees representation</a>).</li>
</ul>
</li>
<li><b>clonal_trees_vertices/</b>
<ul>
<li> clonal_trees_vertices/clonal_tree_1-14-2.tree — a tsv dataframe for each cluster/tree containing all its vertices (details in <a href= "#output_files">Clonal trees representation</a>).</li>
</ul>
</li>
<li><b>cdr3_graphs/ ===========TODO===========</b>
</li>
</ul><br>
<li>Statistics:</li>
<ul>
<li>
<b>cdr_details.txt</b> — tsv file with information about all three CDRs of each clone.
</li>
<li>
<b>shm_details.txt</b> — file with information about all SHMs of each clone.
</li>
<li>
<b>tree_details.txt</b> — tsv file with information about size, depth etc. of all connected trees.
</li>
</ul><br>
<li><b>antevolo.log</b> — full log of AntEvolo run.</li>
</ul>
<br>
<!-- -------------------------------------------------------------------- -->
<!-- -------------------------------------------------------------------- -->
<a id="treevisualizer_usage"></a>
<h2>4. TreeVisualizer usage</h2>
TreeVisualizer takes as an input a directory with AntEvolo results (or any directory containing consistent clonal_trees and clonal_trees_vertices subdirectories). </br></br>
► To run TreeVisualizer, type:
<pre class="code">
<code>
./tree_visualizer.py -i <input_dir> -o <output_dir> -s topk -k <K>
</code>
</pre>
to draw <code><K></code> top-sized trees
</br></br>
<b>OR</b>
</br></br>
<pre class="code">
<code>
./tree_visualizer.py -i <input_dir> -o <output_dir> -s single -n <tree_name>
</code>
</pre>
to draw single tree where <code><tree_name></code> is the name of the needed file within the <code><input_dir>/clonal_trees/</code>
<!-- --------------------- -->
<a id = "visualizer_basic"></a>
<h3>4.1. Basic options:</h3>
<code>-i <input_dir></code></br>
input directory with consistent <code>clonal_trees</code> and <code>clonal_trees_vertices</code> subdirectories (required).</br></br>
<code>-o <output_dir></code></br>
output directory (required).</br></br>
<code>-s <str></code></br>
strategy. Available values are <code>single</code> / <code>topk</code> (required).</br></br>
<code>-k <int></code></br>
number of top-sized trees to draw (required if <code>strategy</code> is <code>topk</code>).</br></br>
<code>-n <str></code></br>
file name of the tree to draw (required if <code>strategy</code> is <code>single</code>).</br></br>
<code>--help, -h</code></br>
Printing help.</br>
<!--- --------------------- -->
<a id="visualizer_examples"></a>
<h3>4.2. Examples</h3>
To draw the clonal tree from file <code>AntEvolo_results_dir/clonal_trees/clonal_tree_14-193-4_Vsize_40_Esize_39.tree</code>, type:
<pre class="code">
<code>
./tree_visualizer.py -i AntEvolo_results_dir -o output_dir -s single -n clonal_tree_14-193-4_Vsize_40_Esize_39.tree
</code>
</pre>
<!-- --------------------- -->
<a id = "visualizer_output"></a>
<h3>4.3. Output files:</h3>
<ul>
<li><tree_name>.dot — file with graph information in <b>dot</b> format</li>
<li><tree_name>.dot.pdf — a pdf image of the tree</li>
</ul>
<!-- -------------------------------------------------------------------- -->
<a id="examples"></a>
<h2>5. Examples</h2>
Example shows IgRepertoireConstructor pipeline in action for merged paired-end Illumina MiSeq library including reads <b>reads.fastq</b> and mass spectra <b>AspN_CID.mzXML</b> corresponding to the same antibody repertoire.<br><br>
► To run IgReC with standard settings, type the following command:
<pre class = "code">
<code>
./igrec.py -s reads.fastq -o repertoire_constructing
</code>
</pre>
Sequences of the constructed repertoire are located in <b>repertoire_constructing/constructed_repertoire.clusters.fa</b>. They can be converted into amino acid sequences and used as a database for matching mass spectra <b>AspN_CID.mzXML</b> (e.g., using MS-GF+ tool). Let result of MS-GF+ tool be a file <b>AspN_CID.mzId</b>.<br><br>
► To run MassSpectraAnalyzer on <b>AspN_CID.mzId</b> file, type the following command:
<pre class = "code">
<code>
./mass_spectra_analyzer.py -o ms_analysis AspN_CID.mzId
</code>
</pre>
Statistics for mass spectra alignment can be found in <b>ms_analysis</b> directory.
<br><br>
<!-- -------------------------------------------------------------------- -->
<a id="output_files"></a>
<h2>6. Antibody repertoire representation</h2>
We used two files for representation of repertoire for the set of clustered reads: CLUSTERS.FASTA and RCM.
<a id="clusters_fasta"></a>
<h3>6.1. CLUSTERS.FASTA file format</h3>
CLUSTERS.FASTA is a FASTA file, where sequences correspond to the assembled antibodies.
Each header contains information about corresponding antibody cluster (id and size):
<pre class="code"> <code>
>cluster___1___size___3
CCCCTGCAATTAAAATTGTTGACCACCTACATACCAAAGACGAGCGCCTTTACGCTTGCCTTTAGTACCTCGCAACGGCTGCGGACG
>cluster___2___size___2
CCCCTGCAATTAAAATTGTTGACCACCTACATACCAAAGACGAGCGCCTTTACGCTTGCCTTTAGTACCTCGCAACGGCTGCGG
>cluster___3___size___1
CCCCTGCAATTAAAATTGTTGACCACCTACATACCAAAGACGAGCGCCTTTACGCTTGCCTTTAGTACCTCGCAACGGCTGCGGAC
</code>
</pre>
<a id="read_cluster_map"></a>
<h3>6.2. RCM file format</h3>
Every line of RCM (read-cluster map) file contains information about read name and corresponding cluster ID:
<pre class="code"> <code>
MISEQ@:53:000000000-A2BMW:1:2114:14345:28882 1
MISEQ@:53:000000000-A2BMW:1:2114:14374:28884 1
MISEQ@:53:000000000-A2BMW:1:2114:14393:28886 1
MISEQ@:53:000000000-A2BMW:1:2114:16454:28882 2
MISEQ@:53:000000000-A2BMW:1:2114:16426:28886 2
MISEQ@:53:000000000-A2BMW:1:2114:15812:28886 3
</code>
</pre>
<br>
Reperoire described in the example above consists of three antibodies. E.g., the antibody with ID 1 has abundancy 3, since it was constructed from three reads:<br>
MISEQ@:53:000000000-A2BMW:1:2114:14345:28882<br>
MISEQ@:53:000000000-A2BMW:1:2114:14374:28884<br>
MISEQ@:53:000000000-A2BMW:1:2114:14393:28886<br><br>
<b>NOTE:</b> IDs in CLUSTERS.FASTA and RCM files are consistent.
<br><br>
<a id = "alignment_info"></a>
<h3 >6.3 Alignment Info file format</h3>
File <b>alignment_info.csv</b> contains the following information about the closest V and J gene segments
in tab-separated view.<br>
Read ids are consistent with headers in file <b>cleaned_reads.fastq</b>.<br>
Ids of V and J gene segments are taken from IMGT database.
<table width = 100%>
<tr align="center">
<td><b>Read id</b></td>
<td><b>V start</b></td>
<td><b>V end</b></td>
<td><b>V score </br>(% identity)</b></td>
<td><b>V id</b></td>
<td><b>J start</b></td>
<td><b>J end</b></td>
<td><b>J score </br>(% identity)</b></td>
<td><b>J id</b></td>
</tr>
<tr align="center">
<td>read1</td> <td>1</td> <td>296</td> <td>100.0</td> <td>IGHV3-20*01</td> <td>321</td>
<td>366</td> <td>89.0</td> <td>IGHJ5*02</td>
</tr>
<tr align="center">
<td>read2</td> <td>1</td> <td>294</td> <td>98.64</td> <td>IGHV3-9*01</td> <td>309</td>
<td>354</td> <td>100.0</td> <td>IGHJ2*01</td>
</tr>
<tr align="center">
<td>...</td> <td>...</td> <td>...</td> <td>...</td> <td>...</td> <td>...</td>
<td>...</td> <td>...</td> <td>...</td>
</tr>
</table>
<!--- -------------------------------------------------------------------- --->
<a id="feedback"></a>
<h2>7. Feedback and bug reports</h2>
Your comments, bug reports, and suggestions are very welcome.
They will help us to further improve AntEvolo.
<br><br>
If you have any trouble running AntEvolo, please send us the log file from the output directory.
<br><br>
Address for communications: <a href="mailto:[email protected]">[email protected]</a>.
<a id = "citation"></a>
<h3>7.1. Citation</h3>
If you use AntEvolo in your research, please refer to
<a href="..."></a>.
</body></html>