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<div class=Section1>
<p class=MsoNormal> </p>
<p class=MsoNormal> </p>
<p class=MsoNormal> </p>
<p class=MsoNormal> </p>
<p class=MsoNormal align=right style='text-align:right'><span style='font-size:
24.0pt;font-family:"Arial Black"'>MUSCLE User Guide</span></p>
<p class=MsoNormal align=right style='text-align:right'><u> </u></p>
<p class=MsoNormal align=right style='text-align:right'><b><span
style='font-family:Arial'> </span></b></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><span style='font-family:Arial'>Multiple sequence comparison
by log-expectation</span></p>
<p class=MsoNormal><span style='font-family:Arial'>by Robert C. Edgar</span></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><span style='font-family:Arial'>Version 3.5</span></p>
<p class=MsoNormal><span style='font-family:Arial'>August 2004</span></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><span style='font-family:Arial'><a
href="http://www.drive5.com/muscle">http://www.drive5.com/muscle</a></span></p>
<p class=MsoNormal><span style='font-family:Arial'>email: muscle (at) drive5.com</span></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><span style='font-family:Arial'>MUSCLE is updated regularly.
Send me an e-mail if you would like to be notified of new releases.</span></p>
<p class=MsoNormal><span style='font-family:Arial'>�</span></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><i><span style='font-family:Arial'>Citation:</span></i></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<p class=MsoNormal><span style='font-family:Arial'><a
href="http://nar.oupjournals.org/cgi/content/full/32/5/1792?ijkey=48Nmt1tta0fMg&keytype=ref">Edgar,
Robert C. (2004), MUSCLE: multiple sequence alignment with high accuracy and
high throughput, <i>Nucleic Acids Research</i> <b>32</b>(5), 1792-97</a>.</span></p>
<span style='font-size:10.0pt;font-family:Arial'><br clear=all
style='page-break-before:always'>
</span>
<p class=MsoNormal><b><span style='font-size:16.0pt;font-family:Arial'>Table of
Contents</span></b></p>
<p class=MsoToc1><span
class=MsoHyperlink><a href="#_Toc81224823">1 Introduction<span
style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>3</span></a></span></p>
<p class=MsoToc1><span class=MsoHyperlink><a href="#_Toc81224824">2 Quick Start<span
style='color:windowtext;display:none;text-decoration:none'> </span><span
style='color:windowtext;display:none;text-decoration:none'>3</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224825">2.1
Installation<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>3</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224826">2.2 Making an
alignment<span style='color:windowtext;display:none;text-decoration:none'> </span><span
style='color:windowtext;display:none;text-decoration:none'>3</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224827">2.3 Large
alignments<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>3</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224828">2.4 Fastest
speed<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>3</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224829">2.5 Huge
alignments<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>4</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224830">2.6 Accuracy:
caveat emptor<span style='color:windowtext;display:none;text-decoration:none'> </span><span
style='color:windowtext;display:none;text-decoration:none'>4</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224831">2.7
Pipelining<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>4</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224832">2.8 Refining
an existing alignment<span style='color:windowtext;display:none;text-decoration:
none'> </span><span
style='color:windowtext;display:none;text-decoration:none'>4</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224833">2.9
Profile-profile alignment<span style='color:windowtext;display:none;text-decoration:
none'> </span><span
style='color:windowtext;display:none;text-decoration:none'>4</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224834">2.10 Sequence
clustering<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>5</span></a></span></p>
<p class=MsoToc1><span class=MsoHyperlink><a href="#_Toc81224835">3 File
Formats<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>5</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224836">3.1 Input
files<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>5</span></a></span></p>
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc81224837">3.1.1 Amino
acid sequences<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>5</span></a></span></p>
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc81224838">3.1.2
Nucleotide sequences<span style='color:windowtext;display:none;text-decoration:
none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>5</span></a></span></p>
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc81224839">3.1.3
Determining sequence type<span style='color:windowtext;display:none;text-decoration:
none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>5</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224840">3.2 Output
files<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>6</span></a></span></p>
<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc81224841">3.2.1
Sequence grouping<span style='color:windowtext;display:none;text-decoration:
none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>6</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224842">3.3 CLUSTALW
format<span style='color:windowtext;display:none;text-decoration:none'> </span><span
style='color:windowtext;display:none;text-decoration:none'>6</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224843">3.4 MSF format<span
style='color:windowtext;display:none;text-decoration:none'> </span><span
style='color:windowtext;display:none;text-decoration:none'>6</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224844">3.5 HTML
format<span style='color:windowtext;display:none;text-decoration:none'> </span><span
style='color:windowtext;display:none;text-decoration:none'>6</span></a></span></p>
<p class=MsoToc1><span class=MsoHyperlink><a href="#_Toc81224845">4 Using
MUSCLE<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>6</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224846">4.1 How the
algorithm works<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>6</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224847">4.2
Command-line options<span style='color:windowtext;display:none;text-decoration:
none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>7</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224848">4.3 The
maxiters option<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>7</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224849">4.4 The
maxtrees option<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>8</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224850">4.5 The
maxhours option<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>8</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224851">4.6 The
profile scoring function<span style='color:windowtext;display:none;text-decoration:
none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>8</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224852">4.7 Diagonal
optimization<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>8</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224853">4.8 Anchor
optimization<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>8</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224854">4.9 Log file<span
style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>8</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224855">4.10 Progress
messages<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>9</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224856">4.11 Running
out of memory<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>9</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224857">4.12
Troubleshooting<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>9</span></a></span></p>
<p class=MsoToc2><span class=MsoHyperlink><a href="#_Toc81224858">4.13
Technical support<span style='color:windowtext;display:none;text-decoration:
none'> </span><span
style='color:windowtext;display:none;text-decoration:none'>10</span></a></span></p>
<p class=MsoToc1><span class=MsoHyperlink><a href="#_Toc81224859">5 Command
Line Reference<span style='color:windowtext;display:none;text-decoration:none'>. </span><span
style='color:windowtext;display:none;text-decoration:none'>10</span></a></span></p>
<p class=MsoNormal><span style='font-family:Arial'> </span></p>
<b><span style='font-size:16.0pt;font-family:Arial'><br clear=all
style='page-break-before:always'>
</span></b>
<h1 style='margin-left:0in;text-indent:0in'><a name="_Toc81224823">1 Introduction</a></h1>
<p class=MsoNormal>MUSCLE is a program for creating multiple alignments of
amino acid or nucleotide sequences. A range of options is provided that give
you the choice of optimizing accuracy, speed, or some compromise between the
two. Default parameters are those that give the best average accuracy in our
tests. Using versions current at the time of writing, my tests show that MUSCLE
can achieve both better average accuracy and better speed than CLUSTALW or T‑Coffee,
depending on the chosen options.</p>
<h1 style='margin-left:0in;text-indent:0in'><a name="_Toc81224824">2 Quick
Start</a></h1>
<p class=MsoNormal>The MUSCLE algorithm is delivered as a command-line program
called <i>muscle</i>. If you are running under Linux or Unix you will be
working at a shell prompt. If you are running under Windows, you should be in a
command window (nostalgically known to us older people as a DOS prompt). If you
don't know how to use command-line programs, you should get help from a local
guru.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224825">2.1 Installation</a></h2>
<p class=MsoNormal>Copy the <i>muscle</i> binary file to a directory that is
accessible from your computer. That's it�there are no configuration files,
libraries, environment variables or other settings to worry about. If you are
using Windows, then the binary file is named <i>muscle.exe</i>. From now on <i>muscle</i>
should be understood to mean "<i>muscle</i> if you are using Linux or Unix,
<i>muscle.exe</i> if you are using Windows".</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224826">2.2 Making an
alignment</a></h2>
<p class=MsoNormal>Make a FASTA file containing some sequences. (If you are not
familiar with FASTA format, it is described in detail later in this Guide.) For
now, just to make things fast, limit the number of sequence in the file to no
more than 50 and the sequence length to be no more than 500. Call the input
file <i>seqs.fa</i>. (An example file named <i>seqs.fa</i> is distributed with
the standard MUSCLE package). Make sure the directory containing the <i>muscle</i>
binary is in your path. (If it isn't, you can run it by typing the full path
name, and the following example command lines must be changed accordingly). Now
type:</p>
<p class=MsoNormal> </p>
<p class=Code>muscle -in seqs.fa -out seqs.afa</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>You should see some progress messages. If <i>muscle</i>
completes successfully, it will create a file <i>seqs.afa</i> containing the
alignment. By default, output is created in "aligned FASTA" format
(hence the <i>.afa</i> extension). This is just like regular FASTA except that
gaps are added in order to align the sequences. This is a nice format for
computers but not very readable for people, so to look at the alignment you
will want an alignment viewer such as Belvu, or a script that converts FASTA to
a more readable format. You can also use the �<i>msf</i> command-line option to
request output in MSF format, which is easier to understand for people. If <i>muscle</i>
gives an error message and you don't know how to fix it, please read the
Troubleshooting section.</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>The default settings are designed to give the best accuracy,
so this may be all you need to know.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224827">2.3 Large
alignments</a></h2>
<p class=MsoNormal>If you have a large number of sequences (a few thousand), or
they are very long, then the default settings of may be too slow for practical
use. A good compromise between speed and accuracy is to run just the first two
iterations of the algorithm. On average, this gives accuracy equal to T-Coffee
and speeds much faster than CLUSTALW. This is done by the option <i>�maxiters 2</i>,
as in the following example.</p>
<p class=MsoNormal> </p>
<p class=Code>muscle -in seqs.fa -out seqs.afa -maxiters 2</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224828">2.4 Fastest
speed</a></h2>
<p class=MsoNormal>If you want the fastest possible speed, then the following
example shows the applicable options for proteins.</p>
<p class=MsoNormal> </p>
<p class=Code>muscle -in seqs.fa -out seqs.afa -maxiters 1 -diags1 -sv -distance1
kbit20_3</p>
<p class=Code> </p>
<p class=MsoNormal>For nucleotides, use:</p>
<p class=MsoNormal> </p>
<p class=Code>muscle -in seqs.fa -out seqs.afa -maxiters 1 -diags1</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>At the time of writing, <i>muscle</i> with these options is faster
than any other multiple sequence alignment program that I have tested. The
alignments are not bad, especially when the sequences are closely related.
However, as you might expect, this blazing speed comes at the cost of the
lowest average accuracy of the options that <i>muscle</i> provides.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224829">2.5 Huge
alignments</a></h2>
<p class=MsoNormal>If you have a <i>very</i> large number of sequences (several
thousand), or they are very long, then the <i>kbit20_3</i> option may cause
problems because it needs a relatively large amount of memory. Better is to use
the default distance measure, which is roughly 2� or 3� slower but needs less
memory, like this:</p>
<p class=MsoNormal> </p>
<p class=Code>muscle -in seqs.fa -out seqs.afa -maxiters 1 -diags1 -sv</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224830">2.6 Accuracy:
caveat emptor</a></h2>
<p class=MsoNormal>Why do I keep using the clumsy phrase "average
accuracy" instead of just saying "accuracy"? That's because the
quality of alignments produced by MUSCLE varies, as do those produced other programs
such as CLUSTALW and T-Coffee. The state of the art leaves plenty of room for
improvement. Sometimes the fastest speed options to <i>muscle</i> give
alignments that are better than T-Coffee, though the reverse will more often be
the case. With challenging sets of sequences, it is a good idea to make several
different alignments using different <i>muscle</i> options and to try other programs
too. Regions where different alignments agree are more believable than regions
where they disagree. </p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224831">2.7 Pipelining</a></h2>
<p class=MsoNormal>Input can be taken from standard input, and output can be
written to standard output. This is the default, so our first example would
also work like this:</p>
<p class=MsoNormal> </p>
<p class=Code>muscle < seqs.fa > seqs.afa</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224832">2.8 Refining
an existing alignment</a></h2>
<p class=MsoNormal>You can ask <i>muscle</i> to try to improve an existing alignment
by using the �<i>refine</i> option. The input file must then be a FASTA file
containing an alignment. All sequences must be of equal length, gaps can be
specified using dots "." or dashes "�". For example:</p>
<p class=MsoNormal> </p>
<p class=Code>muscle -in seqs.afa -out refined.afa -refine</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224833">2.9 Profile-profile
alignment</a></h2>
<p class=MsoNormal>A fundamental step in the MUSCLE algorithm is aligning two
multiple sequence alignments, each of which contain some of the input
sequences. This operation is sometimes called "profile-profile
alignment". If you have two existing alignments of related sequences you
can use the <i>�profile</i> option of MUSCLE to align those two sequences.
Typical usage is:</p>
<p class=MsoNormal> </p>
<p class=Code>muscle -profile -in1 one.afa -in2 two.afa -out both.afa</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>The alignments in <i>one.afa</i> and <i>two.afa</i>, which
must be in aligned FASTA format, are aligned to each other, keeping input
columns intact and inserting columns of gaps where needed. Output is stored in <i>both.afa</i>.</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>MUSCLE does not compute a similarity measure or measure of
statistical significance (such as an E-value), so this option is not useful for
discriminating homologs from unrelated sequences. For this task, I recommend Sadreyev
& Grishin's COMPASS program.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224834">2.10 Sequence
clustering</a></h2>
<p class=MsoNormal>The first stage in MUSCLE is a fast clustering algorithm.
This may be of use in other applications. Typical usage is:</p>
<p class=MsoNormal> </p>
<p class=Code>muscle -cluster -in seqs.fa -tree1 tree.phy</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>The sequences will be clustered, and a tree written to <i>tree.phy</i>.
Options �<i>weight1</i>, �<i>distance1</i>, �<i>cluster1</i> and �<i>root1</i> can
be applied if desired. Note that by default, UPGMA clustering is used. You can
use</p>
<p class=MsoNormal>��<i>neighborjoining</i> if you prefer, but note that this
is substantially slower than UPGMA for large numbers of sequences.</p>
<h1 style='margin-left:0in;text-indent:0in'><a name="_Toc81224835">3 File
Formats</a></h1>
<p class=MsoNormal>MUSCLE uses FASTA format for both input and output. For
output only, it also offers CLUSTALW, MSF and HTML formats using the �<i>clw</i>,
�<i>msf</i> and �<i>html</i> command-line options.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224836">3.1 Input
files</a></h2>
<p class=MsoNormal>Input files must be in FASTA format. These are plain text
files (word processing files such as Word documents are not understood!). Unix,
Windows and DOS text files are supported (end-of-line may be NL or CR NL). There
is no explicit limit on the length of a sequence, however if you are running a
32-bit version of <i>muscle</i> then the maximum will be very roughly 10,000 letters
due to maximum addressable size of tables required in memory. Each sequence starts
with an annotation line, which is recognized by having a greater-than symbol
">" as its first character. There is no limit on the length of an
annotation line (this is new as of version 3.5), and there is no requirement
that the annotation be unique. The sequence itself follows on one or more
subsequent lines, and is terminated either by the next annotation line or by
the end of the file. </p>
<h3 style='margin-left:0in;text-indent:0in'><a name="_Toc81224837">3.1.1 Amino
acid sequences</a></h3>
<p class=MsoNormal>The standard single-letter amino acid alphabet is used. Upper
and lower case is allowed, the case is not significant. The special characters
X, B, Z and U are understood. X means "unknown amino acid", B is D or
N, Z is E or Q. U is understood to be the 21st amino acid Selenocysteine. White
space (spaces, tabs and the end-of-line characters CR and NL) is allowed inside
sequence data. Dots "." and dashes "�" in sequences are
allowed and are discarded unless the input is expected to be aligned (e.g. for
the �<i>refine</i> option). </p>
<h3 style='margin-left:0in;text-indent:0in'><a name="_Toc81224838">3.1.2 Nucleotide
sequences</a></h3>
<p class=MsoNormal>The usual letters A, G, C, T and U stand for nucleotides.
The letters T and U are equivalent as far as MUSCLE is concerned. N is the
wildcard meaning "unknown nucleotide". R means A or G, Y means C or
T/U. Other wildcards, such as those used by RFAM, are not understood in this
version and will be replaced by Ns. If you would like support for other DNA /
RNA alphabets, please let me know.</p>
<h3 style='margin-left:0in;text-indent:0in'><a name="_Toc81224839">3.1.3 Determining
sequence type</a></h3>
<p class=MsoNormal>By default, MUSCLE looks at the first 100 letters in the
input sequence data (excluding gaps). If 95% or more of those letters are valid
nucleotides (AGCTUN), then the file is treated as nucleotides, otherwise as
amino acids. This method almost always guesses correctly, but you can make sure
by specifying the sequence type on the command line. This is done using the �<i>seqtype</i>
option, which can take the following values:</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>������� ��<i>seqtype protein</i>������������������������� Amino
acid</p>
<p class=MsoNormal>������� �s<i>eqtype nucleo</i>������������������������� Nucleotide</p>
<p class=MsoNormal>������� �<i>seqtype auto</i>������������������������������ Automatic
detection (default).</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224840">3.2 Output
files</a></h2>
<p class=MsoNormal>By default, output is also written in FASTA format. All
letters are upper-case and gaps are represented by dashes "�".</p>
<h3 style='margin-left:0in;text-indent:0in'><a name="_Toc81224841">3.2.1 Sequence
grouping</a></h3>
<p class=MsoNormal>By default, MUSCLE re-arranges sequences so that similar
sequences are adjacent in the output file. (This is done by ordering sequences
according to a prefix traversal of the guide tree). This makes the alignment
easier to evaluate by eye. If you want to the sequences to be output in the
same order as the input file, you can use the �<i>stable</i> option.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224842">3.3 CLUSTALW
format</a></h2>
<p class=MsoNormal>You can request CLUSTALW output by using the �<i>clw</i>
option. This should be compatible with CLUSTALW, with the exception of the
program name in the file header. You can ask MUSCLE to impersonate CLUSTALW by
writing "CLUSTAL W (1.81)" as the program name by using �<i>clwstrict</i>.
If you have problems parsing MUSCLE output with scripts designed for CLUSTALW,
please let me know.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224843">3.4 MSF format</a></h2>
<p class=MsoNormal>MSF format (similar to CLUSTALW) is requested by using the �<i>msf</i>
option. As with CLUSTALW format, this is easier for people to read than FASTA.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224844">3.5 HTML
format</a></h2>
<p class=MsoNormal>I've added an experimental feature starting in version 3.4. To
get a Web page as output, use the �<i>html</i> option. The alignment is colored
using a color scheme from Eric Sonnhammer's Belvu editor, which is my personal
favorite. A drawback of this option is that the Web page typically contains a
very large number of HTML tags, which can be slow to display in the Internet
Explorer browser. The Netscape browser works much better. If you have any ideas
about good ways to make Web pages, please let me know.</p>
<h1 style='margin-left:0in;text-indent:0in'><a name="_Toc81224845">4 Using
MUSCLE</a></h1>
<p class=MsoNormal>In this section we give more details of the MUSCLE algorithm
and the more important options offered by the <i>muscle</i> implementation.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224846">4.1 How the
algorithm works</a></h2>
<p class=MsoNormal>We won't give a complete description of the MUSCLE algorithm
here�for that, you will have to read the paper. But hopefully a summary will
help explain what some of the command-line options do and how they might be
useful in your work.</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>The first step is to calculate a tree. In CLUSTALW, this is
done as follows. Each pair of input sequences is aligned, and used to compute
the pair-wise identity of the pair. Identities are converted to a measure of
distance. Finally, the distance matrix is converted to a tree using a
clustering method (CLUSTALW uses neighbor-joining). If you have 1,000
sequences, there are (1,000 <span style='font-family:Symbol'>�</span> 999)/2 =
499,500 pairs, so aligning every pair can take a while. MUSCLE uses a much
faster, but somewhat more approximate, method to compute distances: it counts
the number of short sub-sequences (known as <i>k</i>-mers, <i>k</i>-tuples or
words) that two sequences have in common, without constructing an alignment.
This is typically around 3,000 times faster that CLUSTALW's method, but the trees
will generally be less accurate. We call this step "<i>k</i>-mer
clustering".</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>The second step is to use the tree to construct what is
known as a progressive alignment. At each node of the binary tree, a pair-wise
alignment is constructed, progressing from the leaves towards the root. The
first alignment will be made from two sequences. Later alignments will be one
of the three following types: sequence-sequence, profile-sequence or
profile-profile, where "profile" means the multiple alignment of the sequences
under a given internal node of the tree. This is very similar to what CLUSTALW
does once it has built a tree.</p>
<p class=MsoNormal> </p>
<p class=MsoNormal style='page-break-after:avoid'>Now we have a multiple
alignment, which has been built very quickly compared with conventional methods,
mainly because of the distance calculation using <i>k</i>-mers rather than
alignments. The quality of this alignment is typically pretty good�it will
often tie or beat a T-Coffee alignment on our tests. However, on average, we
find that it can be improved by proceeding through the following steps.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
<p class=MsoNormal style='page-break-after:avoid'>From the multiple alignment,
we can now compute the pair-wise identities of each pair of sequences. This
gives us a new distance matrix, from which we estimate a new tree. We compare
the old and new trees, and re-align subgroups where needed to produce a
progressive multiple alignment from the new tree. If the two trees are
identical, there is nothing to do; if there are no subtrees that agree (very unusual),
then the whole progressive alignment procedure must be repeated from scratch.
Typically we find that the tree is pretty stable near the leaves, but some
re-alignments are needed closer the root. This procedure (compute pair-wise
identities, estimate new tree, compare trees, re-align) is iterated until the
tree stabilizes or until a specified maximum number of iterations has been
done. We call this process "tree refinement", although it also tends
to improve the alignment.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
<p class=MsoNormal style='page-break-after:avoid'>We now keep the tree fixed
and move to a new procedure which is designed to improve the multiple
alignment. The set of sequences is divided into two subsets (i.e., we make a
bipartition on the set of sequences). A profile is constructed for each of the
two subsets based on the current multiple alignment. These two profiles are
then re-aligned to each other using the same pair-wise alignment algorithm as
used in the progressive stage. If this improves an "objective score"
that measures the quality of the alignment, then the new multiple alignment is
kept, otherwise it is discarded. By default, the objective score is the classic
sum-of-pairs score that takes the (sequence weighted) average of the pair-wise
alignment score of every pair of sequences in the alignment. Bipartitions are
chosen by deleting an edge in the guide tree, each of the two resulting
subtrees defines a subset of sequences. This procedure is called "tree
dependent refinement". One iteration of tree dependent refinement tries
bipartitions produced by deleting every edge of the tree in depth order moving
from the leaves towards the center of the tree. Iterations continue until
convergence or up to a specified maximum.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
<p class=MsoNormal style='page-break-after:avoid'>For convenience, the major
steps in MUSCLE are described as "iterations", though the first three
iterations all do quite different things and may take very different lengths of
time to complete. The tree-dependent refinement iterations 3, 4 ... are true
iterations and will take similar lengths of time. </p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0
style='border-collapse:collapse'>
<tr>
<td width=89 valign=top style='width:66.6pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><b><span style='font-size:
9.0pt;font-family:Arial'>Iteration</span></b></p>
</td>
<td width=461 valign=top style='width:4.8in;border:none;border-bottom:solid windowtext 1.0pt;
padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><b><span style='font-size:
9.0pt;font-family:Arial'>Actions</span></b></p>
</td>
</tr>
<tr>
<td width=89 valign=top style='width:66.6pt;border:none;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>1</p>
</td>
<td width=461 valign=top style='width:4.8in;border:none;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Distance matrix by <i>k</i>-mer
clustering, estimate tree, progressive alignment according to this tree.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr>
<td width=89 valign=top style='width:66.6pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>2</p>
</td>
<td width=461 valign=top style='width:4.8in;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Distance matrix by
pair-wise identities from current multiple alignment, estimate tree,
progressive alignment according to new tree, repeat until convergence or specified
maximum number of times.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr>
<td width=89 valign=top style='width:66.6pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>3, 4 ...</p>
</td>
<td width=461 valign=top style='width:4.8in;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Tree-dependent refinement. One
iteration visits every edge in the tree one time.</p>
</td>
</tr>
</table>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224847">4.2 Command-line
options</a></h2>
<p class=MsoNormal>There are two types of command-line options: value options
and flag options. Value options are followed by the value of the given
parameter, for example �<i>in <filename></i>; flag options just stand for
themselves, such as �<i>msf</i>. All options are a dash (not two dashes!)
followed by a long name; there are no single-letter equivalents. Value options
must be separated from their values by white space in the command line. Thus, <i>muscle</i>
does not follow Unix, Linux or Posix standards, for which we apologize. The
order in which options are given is irrelevant unless two options contradict,
in which case the right-most option silently wins.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224848">4.3 The
maxiters option</a></h2>
<p class=MsoNormal>You can control the number of iterations that MUSCLE does by
specifying the <i>�maxiters</i> option. If you specify 1, 2 or 3, then this is
exactly the number of iterations that will be performed. If the value is
greater than 3, then <i>muscle</i> will continue up to the maximum you specify
or until convergence is reached, which ever happens sooner. The default is 16.
If you have a large number of sequences, refinement may be rather slow.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224849">4.4 The maxtrees
option</a></h2>
<p class=MsoNormal>This option controls the maximum number of new trees to
create in iteration 2. Our experience suggests that a point of diminishing
returns is typically reached after the first tree, so the default value is 1.
If a larger value is given, the process will repeat until convergence or until
this number of trees has been created, which ever comes first.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224850">4.5 The maxhours
option</a></h2>
<p class=MsoNormal>If you have a large alignment, <i>muscle</i> may take a long
time to complete. It is sometimes convenient to say "I want the best
alignment I can get in 24 hours" rather than specifying a set of options
that will take an unknown length of time. This is done by using <i>�maxhours</i>,
which specifies a floating-point number of hours. If this time is exceeded, <i>muscle</i>
will write out current alignment and stop. For example,</p>
<p class=MsoNormal> </p>
<p class=Code>muscle -in huge.fa -out huge.afa -maxiters 9999 -maxhours 24.0</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>Note that the actual time may exceed the specified limit by
a few minutes while <i>muscle</i> finishes up on a step. It is also possible
for no alignment to be produced if the time limit is too small.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224851">4.6 The
profile scoring function</a></h2>
<p class=MsoNormal>Three different protein profile scoring functions are
supported, the log-expectation score (�<i>le</i> option) and a sum of pairs
score using either the PAM200 matrix (�<i>sp</i>) or the VTML240 matrix (�<i>sv</i>).
The log-expectation score is the default as it gives better results on our
tests, but is typically somewhere between two or three times slower than the
sum-of-pairs score. For nucleotides, �<i>spn</i> is currently the only option
(which is of course the default for nucleotide data, so you don't need to
specify this option).</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224852">4.7 Diagonal
optimization</a></h2>
<p class=MsoNormal>Creating a pair-wise alignment by dynamic programming
requires computing an <i>L</i><sub>1</sub> <span style='font-family:Symbol'>�</span>
<i>L</i><sub>2</sub> matrix, where <i>L</i><sub>1</sub> and <i>L</i><sub>2</sub>
are the sequence lengths. A trick used in algorithms such as BLAST is to reduce
the size of this matrix by using fast methods to find "diagonals",
i.e. short regions of high similarity between the two sequences. This speeds up
the algorithm at the expense of some reduction in accuracy. MUSCLE uses a
technique called <i>k</i>-mer extension to find diagonals. It is disabled by
default because of the slight reduction in average accuracy and can be turned
on by specifying the �<i>diags1</i> and <i>�diags2</i> options.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224853">4.8 Anchor
optimization</a></h2>
<p class=MsoNormal>Tree-dependent refinement (iterations 3, 4 ... ) can be
speeded up by dividing the alignment vertically into blocks. Block boundaries
are found by identifying high-scoring columns (e.g., a perfectly conserved
column of Cs or Ws would be a candidate). Each vertical block is then refined
independently before reassembling the complete alignment, which is faster
because of the <i>L</i><sup>2</sup> factor in dynamic programming (e.g.,
suppose the alignment is split into two vertical blocks, then 2 <span
style='font-family:Symbol'>�</span> 0.5<sup>2</sup> = 0.5, so the dynamic
programming time is roughly halved). The �<i>noanchors</i> option is used to disable
this feature. This option has no effect if <i>�maxiters 1</i> or �<i>maxiters 2</i>
is specified. On benchmark tests, enabling anchors has little or no effect on
accuracy, but if you want to be very conservative and are striving for the best
possible accuracy then �<i>noanchors</i> is a reasonable choice.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224854">4.9 Log file</a></h2>
<p class=MsoNormal>You can specify a log file by using �<i>log <filename></i>
or �<i>loga <filename></i>. Using �<i>log</i> causes any existing file to
be deleted, �<i>loga</i> appends to any existing file. A message will be
written to the log file when <i>muscle</i> starts and stops. Error and warning
messages will also be written to the log. If �<i>verbose</i> is specified, then
more information will be written, including the command line used to invoke <i>muscle</i>,
the resulting internal parameter settings, and also progress messages. The
content and format of verbose log file output is subject to change in future
versions. </p>
<p class=MsoNormal> </p>
<p class=MsoNormal>The use of a log file may seem contrary to Unix conventions for
using standard output and standard error. I like these conventions, but never
found a fully satisfactory way to use them. I like progress messages (see
below), but they mess up a file if you re-direct standard error and there are
errors or warning messages too. I could try to detect whether a standard file
handle is a <i>tty</i> device or a disk file and change behavior accordingly,
but I regard this as too complicated and too hard for the user to understand. On
Windows it can be hard to re-direct standard file handles, especially when
working in a GUI debugger. Maybe one day I will figure out a better solution
(suggestions welcomed).</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>I highly recommend using �<i>verbose </i>and ��<i>log[a]</i>,
especially when running <i>muscle</i> in a batch mode. This enables you to
verify whether a particular alignment was completed and to review any errors or
warnings that occurred.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224855">4.10 Progress
messages</a></h2>
<p class=MsoNormal>By default, <i>muscle</i> writes progress messages to
standard error periodically so that you know it's doing something and get some
feedback about the time and memory requirements for the alignment. Here is a
typical progress message.</p>
<p class=MsoNormal> </p>
<p class=Code>00:00:23���� 25 Mb� Iter�� 2� 87.20%� Build guide tree</p>
<p class=MsoNormal> </p>
<p class=MsoNormal style='page-break-after:avoid'>The fields are as follows.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0
style='border-collapse:collapse'>
<tr>
<td width=118 valign=top style='width:88.2pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=Code style='page-break-after:avoid'>00:00:23</p>
</td>
<td width=408 valign=top style='width:306.05pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Elapsed time since <i>muscle</i>
started.</p>
</td>
</tr>
<tr>
<td width=118 valign=top style='width:88.2pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=Code style='page-break-after:avoid'>25 Mb</p>
</td>
<td width=408 valign=top style='width:306.05pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Peak memory use in megabytes
(i.e., not the current usage, but the maximum amount of memory used since <i>muscle</i>
started).</p>
</td>
</tr>
<tr>
<td width=118 valign=top style='width:88.2pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=Code style='page-break-after:avoid'>Iter 2</p>
</td>
<td width=408 valign=top style='width:306.05pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Iteration currently in
progress.</p>
</td>
</tr>
<tr>
<td width=118 valign=top style='width:88.2pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=Code style='page-break-after:avoid'>87.20%</p>
</td>
<td width=408 valign=top style='width:306.05pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>How much of the current step
has been completed (percentage).</p>
</td>
</tr>
<tr>
<td width=118 valign=top style='width:88.2pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=Code>Build...</p>
</td>
<td width=408 valign=top style='width:306.05pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>A brief description of the current step.</p>
</td>
</tr>
</table>
<p class=MsoNormal> </p>
<p class=MsoNormal>The �<i>quiet</i> command-line option disables writing
progress messages to standard error. If the �<i>verbose</i> command-line option
is specified, a progress message will be written to the log file when each
iteration completes. So �<i>quiet</i> and �<i>verbose</i> are not
contradictory.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224856">4.11 Running
out of memory</a></h2>
<p class=MsoNormal>The <i>muscle</i> code tries to deal gracefully with
low-memory conditions by using the following technique. A block of "emergency
reserve" memory is allocated when <i>muscle</i> starts. If a later request
to allocate memory fails, this reserve block is made available, and <i>muscle</i>
attempts to save the current alignment. With luck, the reserved memory will be
enough to allow <i>muscle</i> to save the alignment and exit gracefully with an
informative error message.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224857">4.12 Troubleshooting</a></h2>
<p class=MsoNormal>Here is some general advice on what to do if <i>muscle</i>
fails and you don't understand what happened. The code is designed to fail
gracefully with an informative error message when something goes wrong, but
there will no doubt be situations I haven't anticipated (not to mention bugs).</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>Check the MUSCLE web site for updates, bug reports and other
relevant information.</p>
<p class=MsoNormal> </p>
<p class=MsoNormal style='page-break-after:avoid'>������� <a
href="http://www.drive5.com/muscle">http://www.drive5.com/muscle</a></p>
<p class=MsoNormal> </p>
<p class=MsoNormal>Check the input file to make sure it is in valid FASTA format.
Try giving it to another sequence analysis program that can accept large FASTA
files (e.g., the NCBI <i>formatdb</i> utility) to see if you get an informative
error message. Try dividing the file into two halves and using each half
individually as input. If one half fails and the other does not, repeat until
the problem is localized as far as possible.</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>Use �<i>log</i> or �<i>loga</i> and ��<i>verbose</i> and
check the log file to see if there are any messages that give you a hint about the
problem. Look at the peak memory requirements (reported in progress messages)
to see if you may be exceeding the physical or virtual memory capacity of your
computer.</p>
<p class=MsoNormal> </p>
<p class=MsoNormal>If <i>muscle</i> crashes without giving an error message, or
hangs, then you may need to refer to the source code or use a debugger. A
"debug" version, <i>muscled</i>, may be provided. This is built from
the same source code but with the DEBUG macro defined and without compiler
optimizations. This version runs much more slowly (perhaps by a factor of three
or more), but does a lot more internal checking and may be able to catch
something that is going wrong in the code. The ��<i>core</i> option specifies
that <i>muscle</i> should not catch exceptions. When �<i>core</i> is specified,
an exception may result in a debugger trap or a core dump, depending on the
execution environment. The �<i>nocore</i> option has the opposite effect. In <i>muscle</i>,
�<i>nocore</i> is the default, ��<i>core</i> is the default in <i>muscled</i>.</p>
<h2 style='margin-left:0in;text-indent:0in'><a name="_Toc81224858">4.13 Technical
support</a></h2>
<p class=MsoNormal>I am happy to provide support. But I am busy, and am
offering this program at no charge, so I ask you to make a reasonable effort to
figure things out for yourself before contacting me.</p>
<h1 style='margin-left:0in;text-indent:0in'><a name="_Toc81224859">5 Command Line
Reference</a></h1>
<p class=MsoNormal style='page-break-after:avoid'> </p>
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0
style='border-collapse:collapse'>
<thead>
<tr>
<td width=110 valign=top style='width:82.85pt;border:none;border-bottom:
solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><b><span style='font-size:
9.0pt;font-family:Arial'>Value option</span></b></p>
</td>
<td width=113 valign=top style='width:84.8pt;border:none;border-bottom:solid windowtext 1.0pt;
padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><b><span style='font-size:
9.0pt;font-family:Arial'>Legal values</span></b></p>
</td>
<td width=103 valign=top style='width:77.35pt;border:none;border-bottom:
solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><b><span style='font-size:
9.0pt;font-family:Arial'>Default</span></b></p>
</td>
<td width=264 valign=top style='width:197.8pt;border:none;border-bottom:
solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><b><span style='font-size:
9.0pt;font-family:Arial'>Description</span></b></p>
</td>
</tr>
</thead>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>anchorspacing</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Integer</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>32</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Minimum spacing between
anchor columns.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>center</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Floating point</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>[1]</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Center parameter. Should be
negative.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>cluster1</span></p>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>cluster2</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>upgma</span></p>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>upgmb</span></p>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>neighborjoining</span></p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>upgmb</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Clustering method. cluster1
is used in iteration 1 and 2, cluster2 in later iterations.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>diaglength</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Integer</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>24</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Minimum length of diagonal.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>diagmargin</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Integer</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>5</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Discard this many positions
at ends of diagonal.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>distance1</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'> </span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>kmer6_6</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>kmer20_3</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>kmer20_4</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>kbit20_3</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>kmer4_6</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'> </span></p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>Kmer6_6
(amino) or Kmer4_6 (nucleo)</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Distance measure for iteration 1.</p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>distance2</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'> </span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>kmer6_6</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>kmer20_3</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>kmer20_4</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>kbit20_3</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>pctid_kimura</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>pctid_log</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'> </span></p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>pctid_kimura</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Distance measure for iterations 2, 3 ...</p>
<p class=MsoNormal> </p>
<p class=MsoNormal> </p>
<p class=MsoNormal> </p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>gapopen</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Floating point</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>[1]</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>The gap open score. Must be negative.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>hydro</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Integer</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>5</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Window size for determining whether a region is
hydrophobic.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>hydrofactor</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Floating point</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>1.2</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Multiplier for gap open/close penalties in hydrophobic
regions.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>in</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Any file name</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>standard input</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Where to find the input sequences.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>log</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>File name</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>None.</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Log file name (delete existing file).</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>loga</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>File name</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>None.</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Log file name (append to existing file).</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>maxdiagbreak</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Integer</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>1</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Maximum distance between two diagonals that allows them to
merge into one diagonal.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>maxhours</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Floating point</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>None.</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Maximum time to run in hours. The actual time may exceed
the requested limit by a few minutes. Decimals are allowed, so 1.5 means one
hour and 30 minutes.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>maxiters</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Integer 1, 2 ...</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>16</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Maximum number of iterations.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>maxtrees</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Integer</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>1</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Maximum number of new trees to build in iteration 2.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>minbestcolscore</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Floating point </p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>[1]</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Minimum score a column must
have to be an anchor.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>minsmoothscore</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Floating point</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>[1]</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Minimum smoothed score a
column must have to be an anchor.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>objscore</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>sp</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>ps</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>dp</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>xp</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>spf</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>spm</span></p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>spm</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Objective score used by tree dependent refinement.</p>
<p class=MsoNormal>sp=sum-of-pairs score.</p>
<p class=MsoNormal>spf=sum-of-pairs score (dimer approximation)</p>
<p class=MsoNormal>spm=sp for < 100 seqs, otherwise spf</p>
<p class=MsoNormal>dp=dynamic programming score.</p>
<p class=MsoNormal>ps=average profile-sequence score.</p>
<p class=MsoNormal>xp=cross profile score.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>out</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>File name</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>standard output</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Where to write the alignment.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>root1</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>root2</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>pseudo</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>midlongestspan</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>minavgleafdist</span></p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>psuedo</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Method used to root tree; root1 is used in iteration 1 and
2, root2 in later iterations.</p>
<p class=MsoNormal> </p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>seqtype</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>protein</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>nucleo</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>auto</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'> </span></p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>auto</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Sequence type.</p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>smoothscoreceil</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Floating point</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>[1]</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Maximum value of column score for smoothing purposes.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>smoothwindow</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Integer</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>7</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Window used for anchor column smoothing.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>SUEFF</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Floating point value between 0 and 1.</p>
<p class=MsoNormal> </p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>0.1</span></p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Constant used in UPGMB clustering. Determines the relative
fraction of average linkage (SUEFF) vs. nearest-neighbor linkage (1 � SUEFF).<br>
<br>
</p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>tree1</span></p>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>tree2</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>File name</p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>None</p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Save tree produced in first or second iteration to given
file in Newick (Phylip-compatible) format.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr>
<td width=110 valign=top style='width:82.85pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>weight1</span></p>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>weight2</span></p>
</td>
<td width=113 valign=top style='width:84.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>none</span></p>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>henikoff</span></p>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>henikoffpb</span></p>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>gsc</span></p>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>clustalw</span></p>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>threeway</span></p>
</td>
<td width=103 valign=top style='width:77.35pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>clustalw</span></p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
<td width=264 valign=top style='width:197.8pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'>Sequence weighting scheme.</p>
<p class=MsoNormal style='page-break-after:avoid'>weight1 is used in
iterations 1 and 2.</p>
<p class=MsoNormal style='page-break-after:avoid'>weight2 is used for
tree-dependent refinement.</p>
<p class=MsoNormal style='page-break-after:avoid'>none=all sequences have
equal weight.</p>
<p class=MsoNormal style='page-break-after:avoid'>henikoff=Henikoff &
Henikoff weighting scheme.</p>
<p class=MsoNormal style='page-break-after:avoid'>henikoffpb=Modified
Henikoff scheme as used in PSI-BLAST.</p>
<p class=MsoNormal style='page-break-after:avoid'>clustalw=CLUSTALW method.</p>
<p class=MsoNormal style='page-break-after:avoid'>threeway=Gotoh three-way
method.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
</table>
<p class=MsoNormal> </p>
<table class=MsoTableGrid border=0 cellspacing=0 cellpadding=0
style='border-collapse:collapse'>
<thead>
<tr>
<td width=137 valign=top style='width:102.6pt;border:none;border-bottom:
solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><b><span style='font-size:
9.0pt;font-family:Arial'>Flag option</span></b></p>
</td>
<td width=106 valign=top style='width:1.1in;border:none;border-bottom:solid windowtext 1.0pt;
padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><b><span style='font-size:
9.0pt;font-family:Arial'>Set by default?</span></b></p>
</td>
<td width=348 valign=top style='width:261.0pt;border:none;border-bottom:
solid windowtext 1.0pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><b><span style='font-size:
9.0pt;font-family:Arial'>Description</span></b></p>
</td>
</tr>
</thead>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>anchors</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>yes</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>Use anchor optimization in
tree dependent refinement iterations.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>clw</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>Write output in CLUSTALW
format (default is FASTA).</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>clwstrict</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>Write output in CLUSTALW
format with the "CLUSTAL W (1.81)" header rather than the MUSCLE
version. This is useful when a post-processing step is picky about the file
header.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>core</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>yes in muscle,</p>
<p class=MsoNormal style='page-break-after:avoid'>no in muscled.</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>Do not catch exceptions.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>fasta</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>yes</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>Write output in FASTA
format. Alternatives include �<i>clw</i>,</p>
<p class=MsoNormal style='page-break-after:avoid'>�<i>clwstrict, �msf</i> and
�<i>html</i>.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>group</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>yes</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>Group similar sequences
together in the output. This is the default. See also �<i>stable</i>.</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>html</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'>Write output in HTML format
(default is FASTA).</p>
<p class=MsoNormal style='page-break-after:avoid'> </p>
</td>
</tr>
<tr>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>le</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>maybe</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt'>
<p class=MsoNormal>Use log-expectation profile score (VTML240). Alternatives
are to use �<i>sp</i> or <i>�sv</i>. This is the default for amino acid
sequences.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>msf</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Write output in MSF format (default is FASTA).</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>noanchors</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Disable anchor optimization. Default is �<i>anchors</i>.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>nocore</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no in muscle,</p>
<p class=MsoNormal>yes in muscled.</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Catch exceptions and give an error message if possible.</p>
<p class=MsoNormal> </p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>quiet</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Do not display progress messages.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>refine</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Input file is already aligned, skip first two iterations
and begin tree dependent refinement.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>sp</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Use sum-of-pairs protein profile score (PAM200). Default
is �<i>le.</i></p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>spn</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>maybe</p>
<p class=MsoNormal> </p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Use sum-of-pairs nucleotide profile score (BLASTZ
parameters). This is the only option for nucleotides, and is therefore the
default.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal style='page-break-after:avoid'><span style='font-size:
8.0pt;font-family:"Courier New"'>stable</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Preserve input order of sequences in output file. Default
is to group sequences by similarity (�<i>group</i>).</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>sv</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Use sum-of-pairs profile score (VTML240). Default is �<i>le.</i></p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>termgapsfull</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Terminal gaps penalized with full penalty.</p>
<p class=MsoNormal>[1] Not fully supported in this version.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>termgapshalf</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>yes</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Terminal gaps penalized with half penalty.</p>
<p class=MsoNormal>[1] Not fully supported in this version.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>termgapshalflonger</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Terminal gaps penalized with half penalty if gap relative
to </p>
<p class=MsoNormal>longer sequence, otherwise with full penalty.</p>
<p class=MsoNormal>[1] Not fully supported in this version.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>verbose</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Write parameter settings and progress messages to log
file.</p>
<p class=MsoNormal> </p>
</td>
</tr>
<tr style='height:6.25pt'>
<td width=137 valign=top style='width:102.6pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal><span style='font-size:8.0pt;font-family:"Courier New"'>version</span></p>
</td>
<td width=106 valign=top style='width:1.1in;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>no</p>
</td>
<td width=348 valign=top style='width:261.0pt;padding:0in 5.4pt 0in 5.4pt;
height:6.25pt'>
<p class=MsoNormal>Write version string to stdout and exit.</p>
</td>
</tr>
</table>
<p class=MsoNormal><i> </i></p>
<p class=MsoNormal><i>Notes</i></p>
<p class=MsoNormal>[1] Default depends on the profile scoring function. To
determine the default, use �<i>verbose �log</i> and check the log file.</p>
<p class=MsoNormal><u><span style='text-decoration:none'> </span></u></p>
</div>
</body>
</html>
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