1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
|
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Strict//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-strict.dtd">
<html xmlns="http://www.w3.org/1999/xhtml">
<head>
<title>Bowtie: Tutorial</title>
<meta http-equiv="Content-Type" content="text/html; charset=UTF-8" />
<link rel="stylesheet" type="text/css" href="css/style.css" media="screen" />
<meta name="verify-v1" content="YJT1CfXN3kzE9cr+jvNB+Q73lTfHrv8eivoY+xjblc0=" />
</head>
<body>
<div id="wrap">
<!--#include virtual="top.ssi" -->
<div id="main">
<!--#include virtual="rhsidebar.ssi" -->
<div id="leftside">
<table><tr><td cellpadding=7>
<h1> Getting started</h1><br/>
<div id="toc">
<ul>
<li><a href="#algn">Performing alignments</a></li>
<li><a href="#preb">Installing a pre-built index</a></li>
<li><a href="#newi">Building a new index</a></li>
<li><a href="#sam">Finding variations with SAMtools</a></li>
</ul><br/>
</div>
<p>Download and extract the appropriate
<a href="https://sourceforge.net/project/showfiles.php?group_id=236897&package_id=288231">
Bowtie binary release</a> into a fresh directory.
Change to that directory.</p>
<br/>
<h2 id="algn">Performing alignments</h2><br/>
<p>The Bowtie source and binary packages come with a pre-built index
of the <i>E. coli</i> genome,
and a set of 1,000 35-bp reads simulated from that genome. To use
Bowtie to align those reads, issue the following command. If you
get an error message "command not found", try adding a <tt>./</tt>
before the <tt>bowtie</tt>.</p>
<br/>
<blockquote>bowtie e_coli reads/e_coli_1000.fq</blockquote>
<br/>
<p>
The first argument to <tt>bowtie</tt> is the basename of the index for the
genome to be searched. The second argument is the name of a FASTQ file
containing the reads.
</p>
<p>
Depending on your computer, the run might take a few seconds up to
about a minute. You will see bowtie print many lines of output. Each
line is an alignment for a read. The name of the aligned read appears
in the leftmost column. The final line should say <tt>Reported 699
alignments to 1 output stream(s)</tt> or something similar.
</p>
<p>Next, issue this command:</p>
<br/>
<blockquote>bowtie -t e_coli reads/e_coli_1000.fq e_coli.map</blockquote>
<br/>
<p>This run calculates the same alignments as the previous run, but the
alignments are written to <tt>e_coli.map</tt> (the final argument)
rather than to the screen.
Also, the <tt>-t</tt> option
instructs Bowtie to print timing statistics. The output should look something like this:
</p>
<br/>
<blockquote>
Time loading forward index: 00:00:00<br/>
Time loading mirror index: 00:00:00<br/>
Seeded quality full-index search: 00:00:00<br/>
# reads processed: 1000<br/>
# reads with at least one reported alignment: 699 (69.90%)<br/>
# reads that failed to align: 301 (30.10%)<br/>
Reported 699 alignments to 1 output stream(s)<br/>
Time searching: 00:00:00<br/>
Overall time: 00:00:00<br/>
</blockquote>
</p><br/>
<h2 id="preb">Installing a pre-built index</h2><br/>
<p>Download the pre-built <i>S. cerevisiae</i> genome package by
right-clicking the "<i>S. cerevisiae</i>, CYGD" link in the
"Pre-built indexes" section of the right-hand sidebar and
selecting "Save Link As..." or "Save Target As...". All
pre-built indexes are packaged as <tt>.zip</tt> archives, and
the <i>S. cerevisiae</i> archive is named
<tt>s_cerevisiae.ebwt.zip</tt>. When it has finished
downloading, extract the archive into the Bowtie
<tt>indexes</tt> subdirectory using your preferred unzip tool.
The index is now installed.</p>
<p>To test that the index is properly installed, issue this
command from the Bowtie install directory:</p>
<br/>
<blockquote>bowtie -c s_cerevisiae ATTGTAGTTCGAGTAAGTAATGTGGGTTTG</blockquote>
<br/>
<p>This command searches the <i>S. cerevisiae</i> index with a
single read. The <tt>-c</tt> argument instructs Bowtie to
obtain read sequences directly from the command line rather
than from a file. If the index is installed properly, this
command should print a single alignment and then exit.</p>
<p>If you would rather install pre-built indexes somewhere other
than the <tt>indexes</tt> subdirectory of the Bowtie install
directory, simply set the <tt>BOWTIE_INDEXES</tt> environment
variable to point to your preferred directory and extract
indexes there instead.</p>
<br/>
<h2 id="newi">Building a new index</h2><br/>
<p>The pre-built <i>E. coli</i> index included with Bowtie is built
from the sequence
for strain 536, known to cause urinary tract infections. We will
create a new index from the sequence
of <i>E. coli</i> strain O157:H7, a strain known to cause food poisoning.
Download the
<a href="ftp://ftp.ncbi.nlm.nih.gov/genomes/Bacteria/Escherichia_coli_O157_H7_Sakai_uid57781/NC_002127.fna">sequence file by right-clicking this link</a>
and selecting "Save Link As..." or "Save Target As...". The
sequence file is named <tt>NC_002127.fna</tt>. When the
sequence file is finished downloading, move it to the Bowtie
install directory and issue this command:</p>
<br/>
<blockquote>bowtie-build NC_002127.fna e_coli_O157_H7</blockquote>
<br/>
<p>The command should finish quickly, and print several lines of status messages.
When the command has completed, note that the current directory
contains four new files named <tt>e_coli_O157_H7.1.ebwt</tt>,
<tt>e_coli_O157_H7.2.ebwt</tt>,
<tt>e_coli_O157_H7.rev.1.ebwt</tt>, and
<tt>e_coli_O157_H7.rev.2.ebwt</tt>. These files constitute
the index. Move these files to the <tt>indexes</tt>
subdirectory to install it.
<p>To test that the index is properly installed, issue this
command:</p>
<br/>
<blockquote>bowtie -c e_coli_O157_H7 GCGTGAGCTATGAGAAAGCGCCACGCTTCC</blockquote>
<br/>
<p>If the index is installed properly, this
command should print a single alignment and then exit.</p>
<br/>
<h2 id="sam">Finding variations with SAMtools</h2><br/>
<p><a href="http://samtools.sf.net">SAMtools</a> is a suite of tools for storing,
manipulating, and analyzing alignments such as those output by Bowtie.
SAMtools understands alignments in either of two complementary
formats: the human-readable SAM format, or the binary BAM format.
Because Bowtie can output SAM (using the <tt>-S/--sam</tt> option), and SAM can
can be converted to BAM using SAMtools, Bowtie users can make full use
of the analyses implemented in SAMtools, or in any other tools
supporting SAM or BAM.
</p>
<p>
We will use SAMtools to find SNPs in a set of simulated reads included
with Bowtie. The reads cover the first 10,000 bases of the pre-built
E. coli genome and contain 10 SNPs throughout. First, we run <tt>bowtie</tt>
to align the reads, being sure to specify the <tt>-S</tt> option. We also
specify an output file that we will use as input for the next step
(though pipes can be used to accomplish the same thing without the
intermediate file):
</p>
<br/>
<blockquote>
bowtie -S e_coli reads/e_coli_10000snp.fq ec_snp.sam
</blockquote>
<br/>
<p>
Next, we convert the SAM file to BAM in preparation for sorting. We
assume that SAMtools is installed and that the samtools binary is
accessible in the PATH.
</p>
<br/>
<blockquote>
samtools view -bS -o ec_snp.bam ec_snp.sam
</blockquote>
<br/>
<p>
Next, we sort the BAM file, in preparation for SNP calling:
</p>
<br/>
<blockquote>
samtools sort ec_snp.bam ec_snp.sorted
</blockquote>
<br/>
<p>
We now have a sorted BAM file called <tt>ec_snp.sorted.bam</tt>. Sorted BAM is
a useful format because the alignments are both compressed, which is
convenient for long-term storage, and sorted, which is conveneint for
variant discovery. Finally, we call variants from the Sorted BAM:
</p>
<br/>
<blockquote>
samtools pileup -cv -f genomes/NC_008253.fna ec_snp.sorted.bam
</blockquote>
<br/>
<p>
For this sample data, the <tt>samtools pileup</tt> command should print
records for 10 distinct SNPs, the first being at position 541 in the
reference.
</p>
<p>
See the <a href="http://samtools.sf.net/">SAMtools web site</a> for details
on how to use these and other
tools in the SAMtools suite.
</p>
</td></tr></table>
</div>
</div>
<!--#include virtual="foot.ssi" -->
</div>
<!-- Google analytics code -->
<script type="text/javascript">
var gaJsHost = (("https:" == document.location.protocol) ? "https://ssl." : "http://www.");
document.write(unescape("%3Cscript src='" + gaJsHost + "google-analytics.com/ga.js' type='text/javascript'%3E%3C/script%3E"));
</script>
<script type="text/javascript">
var pageTracker = _gat._getTracker("UA-5334290-1");
pageTracker._trackPageview();
</script>
<!-- End google analytics code -->
</body>
</html>
|
