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<body>
<div class="document" id="lastal">
<h1 class="title">lastal</h1>

<p>This program finds local alignments between query sequences, and
reference sequences that have been prepared using lastdb.  You can use
it like this:</p>
<pre class="literal-block">
lastdb humanDb humanChromosome*.fasta
lastal humanDb dna*.fasta &gt; myalns.maf
</pre>
<p>The lastdb command reads files called <tt class="docutils literal"><span class="pre">humanChromosome*.fasta</span></tt> and
writes several files whose names begin with <tt class="docutils literal">humanDb</tt>.  The lastal
command reads files called <tt class="docutils literal"><span class="pre">dna*.fasta</span></tt>, compares them to
<tt class="docutils literal">humanDb</tt>, and writes alignments to a file called <tt class="docutils literal">myalns.maf</tt>.</p>
<p>You can use gzip (.gz) compressed query files.  You can also pipe
query sequences into lastal, for example:</p>
<pre class="literal-block">
bzcat seqs.fasta.bz2 | lastal humanDb &gt; myalns.maf
</pre>
<div class="section" id="steps-in-lastal">
<h2>Steps in lastal</h2>
<ol class="arabic">
<li><p class="first">Find initial matches.  For each possible start position in the
query: find the shortest match with length ≥ l that <em>either</em> occurs
≤ m times in the reference, <em>or</em> has length L.</p>
</li>
<li><p class="first">Extend a gapless alignment from each initial match, and keep those
with score ≥ d.</p>
</li>
<li><p class="first">Define cores: find the longest run of identical matches in each
gapless alignment.</p>
</li>
<li><p class="first">Extend a gapped alignment from either side of each core, and keep
those with score ≥ e.</p>
</li>
<li><p class="first">Non-redundantize the alignments: if several alignments share an
endpoint (same coordinates in both sequences), remove all but one
highest-scoring one.</p>
</li>
<li><p class="first">Estimate the ambiguity of each aligned column (OFF by default).</p>
</li>
<li><p class="first">Redo the alignments to minimize column ambiguity, using either
gamma-centroid or LAMA (OFF by default).</p>
</li>
</ol>
</div>
<div class="section" id="options">
<h2>Options</h2>
<div class="section" id="cosmetic-options">
<h3>Cosmetic options</h3>
<blockquote>
<table class="docutils option-list" frame="void" rules="none">
<col class="option" />
<col class="description" />
<tbody valign="top">
<tr><td class="option-group">
<kbd><span class="option">-h</span>, <span class="option">--help</span></kbd></td>
<td>Show all options and their default settings, and exit.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-V</span>, <span class="option">--version</span></kbd></td>
<td>Show version information, and exit.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-v</span></kbd></td>
<td>Be verbose: write messages about what lastal is doing.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-f <var>NAME</var></span></kbd></td>
<td><p class="first">Choose the output format.  The NAME is not case-sensitive.</p>
<p><strong>MAF</strong> format looks like this:</p>
<pre class="literal-block">
a score=15 EG2=4.7e+04 E=2.6e-05
s chr3 9 23 + 939557 TTTGGGAGTTGAAGTTTTCGCCC
s seqA 2 21 +     25 TTTGGGAGTTGAAGGTT--GCCC
</pre>
<p>Lines starting with &quot;s&quot; contain: the sequence name, the start
coordinate of the alignment, the number of sequence letters
spanned by the alignment, the strand, the sequence length, and
the aligned letters.  The start coordinates are zero-based.  If
the strand is &quot;-&quot;, the start coordinate is in the reverse
strand.</p>
<p>The same alignment in <strong>TAB</strong> format looks like this:</p>
<pre class="literal-block">
15 chr3 9 23 + 939557 seqA 2 21 + 25 17,2:0,4 EG2=4.7e+04 E=2.6e-05
</pre>
<p>The &quot;17,2:0,4&quot; shows the sizes and offsets of gapless blocks in
the alignment.  In this case, we have a block of size 17, then
an offset of size 2 in the upper sequence and 0 in the lower
sequence, then a block of size 4.</p>
<p>The same alignment in <strong>BlastTab</strong> format looks like this:</p>
<pre class="literal-block">
seqA chr3 86.96 23 1 1 3 23 10 32 2.6e-05 44.3
</pre>
<p>The fields are: query name, reference name, percent identity,
alignment length, mismatches, gap opens, query start, query end,
reference start, reference end, E-value, bit score.  The start
coordinates are one-based.  <em>Warning:</em> this is a lossy format,
because it does not show gap positions.  <em>Warning:</em> the other
LAST programs cannot read this format.  <em>Warning:</em> <a class="reference external" href="last-evalues.html">&quot;bit score&quot;
is not the same as &quot;score&quot;</a>.</p>
<p><strong>BlastTab+</strong> format is the same as BlastTab, with 3 extra
columns at the end: length of query sequence, length of
reference sequence, and (raw) score.  More columns might be
added in future.</p>
<p class="last">For backwards compatibility, a NAME of 0 means TAB and 1 means
MAF.</p>
</td></tr>
</tbody>
</table>
</blockquote>
</div>
<div class="section" id="e-value-options">
<h3>E-value options</h3>
<blockquote>
<table class="docutils option-list" frame="void" rules="none">
<col class="option" />
<col class="description" />
<tbody valign="top">
<tr><td class="option-group">
<kbd><span class="option">-D <var>LENGTH</var></span></kbd></td>
<td>Report alignments that are expected by chance at most once per
LENGTH query letters.  This option only affects the default
value of -E, so if you specify -E then -D has no effect.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-E <var>THRESHOLD</var></span></kbd></td>
<td>Maximum EG2 (<a class="reference external" href="last-evalues.html">expected alignments per square giga</a>).  This option only affects the default
value of -e, so if you specify -e then -E has no effect.</td></tr>
</tbody>
</table>
</blockquote>
</div>
<div class="section" id="score-options">
<h3>Score options</h3>
<blockquote>
<table class="docutils option-list" frame="void" rules="none">
<col class="option" />
<col class="description" />
<tbody valign="top">
<tr><td class="option-group">
<kbd><span class="option">-r <var>SCORE</var></span></kbd></td>
<td>Match score.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-q <var>COST</var></span></kbd></td>
<td>Mismatch cost.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-p <var>NAME</var></span></kbd></td>
<td><p class="first">Specify a match/mismatch score matrix.  Options -r and -q will
be ignored.  The built-in matrices are described in
<a class="reference external" href="last-matrices.html">last-matrices.html</a>.</p>
<p>Any other NAME is assumed to be a file name.  For an example of
the format, see the matrix files in the data directory.
Asymmetric scores are allowed: query letters correspond to
columns and reference letters correspond to rows.</p>
<p>Any letters that aren't in the matrix get default match/mismatch
scores.  For doubly- and triply-ambiguous bases (such as &quot;W&quot;
meaning A or T), these default scores are derived from the ACGT
scores, and are shown in the header of lastal's output.  Any
other letters get the lowest score in the matrix when aligned to
anything.</p>
<p class="last">Other options can be specified on lines starting with &quot;#last&quot;,
but command line options override them.</p>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-a <var>COST</var></span></kbd></td>
<td>Gap existence cost.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-b <var>COST</var></span></kbd></td>
<td>Gap extension cost.  A gap of size k costs: a + (b × k).</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-A <var>COST</var></span></kbd></td>
<td>Insertion existence cost.  This refers to insertions in the
query relative to the reference.  If -A is not used, the
insertion existence cost will equal the deletion existence cost,
which is set by -a.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-B <var>COST</var></span></kbd></td>
<td>Insertion extension cost.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-c <var>COST</var></span></kbd></td>
<td>This option allows use of &quot;generalized affine gap costs&quot; (SF
Altschul 1998, Proteins 32(1):88-96).  Here, a &quot;gap&quot; may consist
of unaligned regions of both sequences.  If these unaligned
regions have sizes j and k, where j ≤ k, the cost is: a +
b⋅(k-j) + c⋅j.  If c ≥ a + 2b (the default), it reduces to
standard affine gaps.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-F <var>COST</var></span></kbd></td>
<td><p class="first">Align DNA queries to protein reference sequences, using the
specified frameshift cost.  A value of 15 seems to be
reasonable.  (As a special case, -F0 means DNA-versus-protein
alignment without frameshifts, which is faster.)  The output
looks like this:</p>
<pre class="literal-block">
a score=108
s prot 2  40 + 649 FLLQAVKLQDP-STPHQIVPSP-VSDLIATHTLCPRMKYQDD
s dna  8 117 + 999 FFLQ-IKLWDP\STPH*IVSSP/PSDLISAHTLCPRMKSQDN
</pre>
<p>The <tt class="docutils literal">\</tt> indicates a forward shift by one nucleotide, and the
<tt class="docutils literal">/</tt> indicates a reverse shift by one nucleotide.  The <tt class="docutils literal">*</tt>
indicates a stop codon.  The same alignment in tabular format
looks like this:</p>
<pre class="literal-block">
108 prot 2 40 + 649 dna 8 117 + 999 4,1:0,6,0:1,10,0:-1,19
</pre>
<p class="last">The &quot;-1&quot; indicates the reverse frameshift.</p>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-z <var>DROP</var></span></kbd></td>
<td><p class="first">Maximum score drop for gapped alignments.  Gapped alignments are
forbidden from having any internal region with score &lt; -DROP.
The default value is e-1, which arguably produces the best
alignments.  Lower values improve speed, by quitting unpromising
extensions sooner.  You can specify this parameter in 3 ways:</p>
<ul class="last">
<li><p class="first">A score (e.g. -z20).</p>
</li>
<li><p class="first">A percentage.  For example, -z50% specifies 50% of the default
value (rounded down to the nearest integer).</p>
</li>
<li><p class="first">A maximum gap length.  For example, -z8g sets the maximum
score drop to: min[a+8b, A+8B].  However, this never increases
the value above the default.</p>
</li>
</ul>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-x <var>DROP</var></span></kbd></td>
<td>This option makes lastal extend gapped alignments twice.  First,
it extends gapped alignments with a maximum score drop of x, and
discards those with score &lt; e.  The surviving alignments are
redone with a (presumably higher) maximum score drop of z.  This
aims to improve speed with minimal effect on the final
alignments.  You can specify -x in the same ways as -z (with the
default value of x being z).</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-y <var>DROP</var></span></kbd></td>
<td>Maximum score drop for gapless alignments.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-d <var>SCORE</var></span></kbd></td>
<td>Minimum score for gapless alignments.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-e <var>SCORE</var></span></kbd></td>
<td>Minimum alignment score.  (If you do gapless alignment with
option -j1, then -d and -e mean the same thing.  If you set
both, -e will prevail.)</td></tr>
</tbody>
</table>
</blockquote>
</div>
<div class="section" id="initial-match-options">
<h3>Initial-match options</h3>
<blockquote>
<table class="docutils option-list" frame="void" rules="none">
<col class="option" />
<col class="description" />
<tbody valign="top">
<tr><td class="option-group">
<kbd><span class="option">-m <var>MULTIPLICITY</var></span></kbd></td>
<td><p class="first">Maximum multiplicity for initial matches.  Each initial match is
lengthened until it occurs at most this many times in the
reference.</p>
<p class="last">If the reference was split into volumes by lastdb, then lastal
uses one volume at a time.  The maximum multiplicity then
applies to each volume, not the whole reference.  This is why
voluming changes the results.</p>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-l <var>LENGTH</var></span></kbd></td>
<td>Minimum length for initial matches.  Length means the number of
letters spanned by the match.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-L <var>LENGTH</var></span></kbd></td>
<td>Maximum length for initial matches.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-k <var>STEP</var></span></kbd></td>
<td>Look for initial matches starting only at every STEP-th position
in each query (positions 0, STEP, 2×STEP, etc).  This makes
lastal faster but less sensitive.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-W <var>SIZE</var></span></kbd></td>
<td>Look for initial matches starting only at query positions that
are &quot;minimum&quot; in any window of SIZE consecutive positions (see
<a class="reference external" href="lastdb.html">lastdb.html</a>).  By default, this parameter takes the same
value as was used for lastdb -W.</td></tr>
</tbody>
</table>
</blockquote>
</div>
<div class="section" id="miscellaneous-options">
<h3>Miscellaneous options</h3>
<blockquote>
<table class="docutils option-list" frame="void" rules="none">
<col class="option" />
<col class="description" />
<tbody valign="top">
<tr><td class="option-group">
<kbd><span class="option">-s <var>STRAND</var></span></kbd></td>
<td>Specify which query strand should be used: 0 means reverse only,
1 means forward only, and 2 means both.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-S <var>NUMBER</var></span></kbd></td>
<td>Specify how to use the substitution score matrix for reverse
strands.  This matters only for unusual matrices that lack
strand symmetry (e.g. if the a:g score differs from the t:c
score).  &quot;0&quot; means that the matrix is used as-is for all
alignments.  &quot;1&quot; means that the matrix is used as-is for
alignments of query sequence forward strands, and the
complemented matrix is used for query sequence reverse strands.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-K <var>LIMIT</var></span></kbd></td>
<td>Omit any alignment whose query range lies in LIMIT or more other
alignments with higher score (and on the same strand).  This is
a useful way to get just the top few hits to each part of each
query (P Berman et al. 2000, J Comput Biol 7:293-302).</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-C <var>LIMIT</var></span></kbd></td>
<td>Before extending gapped alignments, discard any gapless
alignment whose query range lies in LIMIT or more others (for
the same strand and volume) with higher score-per-length.  This
can reduce run time and output size (MC Frith &amp; R Kawaguchi
2015, Genome Biol 16:106).</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-P <var>THREADS</var></span></kbd></td>
<td>Divide the work between this number of threads running in
parallel.  0 means use as many threads as your computer claims
it can handle simultaneously.  Single query sequences are not
divided between threads, so you need multiple queries per batch
for this option to take effect.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-i <var>BYTES</var></span></kbd></td>
<td><p class="first">Search queries in batches of at most this many bytes.  If a
single sequence exceeds this amount, however, it is not split.
You can use suffixes K, M, and G to specify KibiBytes,
MebiBytes, and GibiBytes.  This option has no effect on the
results (apart from their order).</p>
<p class="last">If the reference was split into volumes by lastdb, then each
volume will be read into memory once per query batch.</p>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-M</span></kbd></td>
<td><p class="first">Find minimum-difference alignments, which is faster but cruder.
This treats all matches the same, and minimizes the number of
differences (mismatches plus gaps).</p>
<ul class="last">
<li><p class="first">Any substitution score matrix will be ignored.  The
substitution scores are set by the match score (r) and the
mismatch cost (q).</p>
</li>
<li><p class="first">The gap cost parameters will be ignored.  The gap existence
cost will be 0 and the gap extension cost will be q + r/2.</p>
</li>
<li><p class="first">The match score (r) must be an even number.</p>
</li>
<li><p class="first">Any sequence quality data (e.g. fastq) will be ignored.</p>
</li>
</ul>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-T <var>NUMBER</var></span></kbd></td>
<td><p class="first">Type of alignment: 0 means &quot;local alignment&quot; and 1 means
&quot;overlap alignment&quot;.  Local alignments can end anywhere in the
middle or at the ends of the sequences.  Overlap alignments must
extend to the left until they hit the end of a sequence (either
query or reference), and to the right until they hit the end of
a sequence.</p>
<p class="last"><strong>Warning:</strong> it's often a bad idea to use -T1.  This setting
does not change the maximum score drop allowed inside
alignments, so if an alignment cannot be extended to the end of
a sequence without exceeding this drop, it will be discarded.</p>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-n <var>COUNT</var></span></kbd></td>
<td>Maximum number of gapless alignments per query position.  When
lastal extends gapless alignments from initial matches that
start at one query position, if it gets COUNT successful
extensions, it skips any remaining initial matches starting at
that position.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-N <var>COUNT</var></span></kbd></td>
<td>Stop after finding COUNT alignments per query strand.  This
makes lastal faster: it quits gapless and gapped extensions as
soon as it finds COUNT alignments with score ≥ e.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-R <var>DIGITS</var></span></kbd></td>
<td><p class="first">Specify lowercase-marking of repeats, by two digits (e.g. &quot;-R 01&quot;),
with the following meanings.</p>
<p>First digit:</p>
<ol class="arabic" start="0">
<li><p class="first">Convert the input sequences to uppercase while reading them.</p>
</li>
<li><p class="first">Keep any lowercase in the input sequences.</p>
</li>
</ol>
<p>Second digit:</p>
<ol class="arabic" start="0">
<li><p class="first">Do not check for simple repeats.</p>
</li>
<li><p class="first">Convert simple repeats (e.g. cacacacacacacacac) to lowercase.</p>
</li>
<li><p class="first">Convert simple repeats, within AT-rich DNA, to lowercase.</p>
</li>
</ol>
<p class="last">Details: Tantan is applied separately to forward and reverse
strands.  For DNA-versus-protein alignment (option -F), it is
applied to the DNA after translation, at the protein level.</p>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-u <var>NUMBER</var></span></kbd></td>
<td><p class="first">Specify treatment of lowercase letters when extending
alignments:</p>
<ol class="arabic" start="0">
<li><p class="first">Mask them for neither gapless nor gapped extensions.</p>
</li>
<li><p class="first">Mask them for gapless but not gapped extensions.</p>
</li>
<li><p class="first">Mask them for gapless but not gapped extensions, and then
discard alignments that lack any segment with score ≥ e when
lowercase is masked.</p>
</li>
<li><p class="first">Mask them for gapless and gapped extensions.</p>
</li>
</ol>
<p>&quot;Mask&quot; means change their match/mismatch scores to min(unmasked
score, 0), a.k.a. <a class="reference external" href="https://doi.org/10.1371/journal.pone.0028819">gentle masking</a>.</p>
<p class="last">This option does not affect treatment of lowercase for initial
matches.</p>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-w <var>DISTANCE</var></span></kbd></td>
<td><p class="first">This option is a kludge to avoid catastrophic time and memory
usage when self-comparing a large sequence.  If the sequence
contains a tandem repeat, we may get a gapless alignment that is
slightly offset from the main self-alignment.  In that case, the
gapped extension might &quot;discover&quot; the main self-alignment and
extend over the entire length of the sequence.</p>
<p>To avoid this problem, gapped alignments are not triggered from
any gapless alignment that:</p>
<ul>
<li><p class="first">is contained, in both sequences, in the &quot;core&quot; of another
alignment</p>
</li>
<li><p class="first">has start coordinates offset by DISTANCE or less relative to
this core</p>
</li>
</ul>
<p class="last">Use -w0 to turn this off.</p>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-G <var>FILE</var></span></kbd></td>
<td>Use an alternative genetic code in the specified file.  For an
example of the format, see vertebrateMito.gc in the examples
directory.  By default, the standard genetic code is used.  This
option has no effect unless DNA-versus-protein alignment is
selected with option -F.</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-t <var>TEMPERATURE</var></span></kbd></td>
<td>Parameter for converting between scores and likelihood ratios.
This affects the column ambiguity estimates.  A score is
converted to a likelihood ratio by this formula: exp(score /
TEMPERATURE).  The default value is 1/lambda, where lambda is
the scale factor of the scoring matrix, which is calculated by
the method of Yu and Altschul (YK Yu et al. 2003, PNAS
100(26):15688-93).</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-g <var>GAMMA</var></span></kbd></td>
<td><p class="first">This option affects gamma-centroid and LAMA alignment only.</p>
<p>Gamma-centroid alignments minimize the ambiguity of paired
letters.  In fact, this method aligns letters whose column error
probability is less than GAMMA/(GAMMA+1).  When GAMMA is low, it
aligns confidently-paired letters only, so there tend to be many
unaligned letters.  When GAMMA is high, it aligns letters more
liberally.</p>
<p>LAMA (Local Alignment Metric Accuracy) alignments minimize the
ambiguity of columns (both paired letters and gap columns).
When GAMMA is low, this method produces shorter alignments with
more-confident columns, and when GAMMA is high it produces
longer alignments including less-confident columns.</p>
<p>In summary: to get the most accurately paired letters, use
gamma-centroid.  To get accurately placed gaps, use LAMA.</p>
<p class="last">Note that the reported alignment score is that of the ordinary
gapped alignment before realigning with gamma-centroid or LAMA.</p>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-j <var>NUMBER</var></span></kbd></td>
<td><p class="first">Output type: 0 means counts of initial matches (of all lengths);
1 means gapless alignments; 2 means gapped alignments before
non-redundantization; 3 means gapped alignments after
non-redundantization; 4 means alignments with ambiguity
estimates; 5 means gamma-centroid alignments; 6 means LAMA
alignments; 7 means alignments with expected counts.</p>
<p>If you use -j0, lastal will count the number of initial matches,
per length, per query sequence.  Options -l and -L will set the
minimum and maximum lengths, and -m will be ignored.  If you
compare a large sequence to itself with -j0, it's wise to set
option -L.</p>
<p>If you use j&gt;3, each alignment will get a &quot;fullScore&quot; (also
known as &quot;forward score&quot; or &quot;sum-of-paths score&quot;).  This is like
the score, but it takes into account alternative alignments.</p>
<p>If you use -j7, lastal will print an extra MAF line starting
with &quot;c&quot; for each alignment.  The first 16 numbers on this line
are the expected counts of matches and mismatches: first the
count of reference As aligned to query As, then the count of
reference As aligned to query Cs, and so on.  For proteins there
will be 400 such numbers.  The final ten numbers are expected
counts related to gaps.  They are:</p>
<ul>
<li><p class="first">The count of matches plus mismatches.  (This may exceed the
total of the preceding numbers, if the sequences have non-ACGT
letters.)</p>
</li>
<li><p class="first">The count of deleted letters.</p>
</li>
<li><p class="first">The count of inserted letters.</p>
</li>
<li><p class="first">The count of delete opens (= count of delete closes).</p>
</li>
<li><p class="first">The count of insert opens (= count of insert closes).</p>
</li>
<li><p class="first">The count of adjacent pairs of insertions and deletions.</p>
</li>
</ul>
<p>The final four numbers are always zero, unless you use
generalized affine gap costs.  They are:</p>
<ul class="last">
<li><p class="first">The count of unaligned letter pairs.</p>
</li>
<li><p class="first">The count of unaligned letter pair opens (= count of closes).</p>
</li>
<li><p class="first">The count of adjacent pairs of deletions and unaligned letter pairs.</p>
</li>
<li><p class="first">The count of adjacent pairs of insertions and unaligned letter pairs.</p>
</li>
</ul>
</td></tr>
<tr><td class="option-group">
<kbd><span class="option">-Q <var>NUMBER</var></span></kbd></td>
<td><p class="first">Specify how to read the query sequences:</p>
<pre class="literal-block">
Default    fasta
   0       fasta or fastq-ignore
   1       fastq-sanger
   2       fastq-solexa
   3       fastq-illumina
   4       prb
   5       PSSM
</pre>
<p><em>Warning</em>: Illumina data is not necessarily in fastq-illumina
format; it is often in fastq-sanger format.</p>
<p>The fastq formats look like this:</p>
<pre class="literal-block">
&#64;mySequenceName
TTTTTTTTGCCTCGGGCCTGAGTTCTTAGCCGCG
+
55555555*&amp;5-/55*5//5(55,5#&amp;$)$)*+$
</pre>
<p>The &quot;+&quot; may be followed by text (ignored).  The symbols below
the &quot;+&quot; are quality codes, one per sequence letter.  The
sequence and quality codes may wrap onto more than one line.</p>
<p>fastq-ignore means that the quality codes are ignored.  For the
other fastq variants, lastal assumes the quality codes indicate
substitution error probabilities, <em>not</em> insertion or deletion
error probabilities.  If this assumption is dubious (e.g. for
data with many insertion or deletion errors), it may be better
to use fastq-ignore.</p>
<p>For fastq-sanger, quality scores are obtained by subtracting 33
from the ASCII values of the quality codes.  For fastq-solexa
and fastq-illumina, they are obtained by subtracting 64.</p>
<p>prb format stores four quality scores (A, C, G, T) per position,
with one sequence per line, like this:</p>
<pre class="literal-block">
-40  40 -40 -40      -12   1 -12  -3      -10  10 -40 -40
</pre>
<p>Since prb does not store sequence names, lastal uses the line
number (starting from 1) as the name.</p>
<p>In fastq-sanger and fastq-illumina format, the quality scores
are related to error probabilities like this: qScore =
-10⋅log10[p].  In fastq-solexa and prb, however, qScore =
-10⋅log10[p/(1-p)].  In lastal's MAF output, the quality scores
are written on lines starting with &quot;q&quot;.  For fastq, they are
written with the same encoding as the input.  For prb, they are
written in the fastq-solexa (ASCII-64) encoding.</p>
<p>Finally, PSSM means &quot;position-specific scoring matrix&quot;.  The
format is:</p>
<pre class="literal-block">
myLovelyPSSM
     A  R  N  D  C  Q  E  G  H  I  L  K  M  F  P  S  T  W  Y  V
1 M -2 -2 -3 -4 -2 -1 -3 -3 -2  1  2 -2  8 -1 -3 -2 -1 -2 -2  0
2 S  0 -2  0  1  3 -1 -1 -1 -2 -3 -3 -1 -2 -3 -2  5  0 -4 -3 -2
3 D -1 -2  0  7 -4 -1  1 -2 -2 -4 -4 -2 -4 -4 -2 -1 -2 -5 -4 -4
</pre>
<p>The sequence appears in the second column, and columns 3 onwards
contain the position-specific scores.  Any letters not specified
by any column will get the lowest score in each row.  This
format is a simplified version of PSI-BLAST's ASCII format: the
non-simplified version is allowed too.</p>
<p class="last"><em>Warning</em>: lastal cannot directly calculate E-values for PSSMs.
The E-values (and the default value of -y) are determined by the
otherwise-unused match and mismatch scores (options -r -q and
-p).  There is evidence these E-values will be accurate if the
PSSM is &quot;constructed to the same scale&quot; as the match/mismatch
scores (SF Altschul et al. 1997, NAR 25(17):3389-402).</p>
</td></tr>
</tbody>
</table>
</blockquote>
</div>
</div>
<div class="section" id="parallel-processes-and-memory-sharing">
<h2>Parallel processes and memory sharing</h2>
<p>If you run several lastal commands (i.e. processes) at the same time
on the same computer, using the same set of reference files prepared
by lastdb, then they will share memory for the reference files.</p>
</div>
<div class="section" id="multiple-volumes">
<h2>Multiple volumes</h2>
<p>If lastdb creates multiple volumes:</p>
<pre class="literal-block">
lastdb hugeDb huge.fasta
</pre>
<p>You can either run lastal on the whole thing:</p>
<pre class="literal-block">
lastal hugeDb queries.fasta &gt; myalns.maf
</pre>
<p>Or on one volume at a time:</p>
<pre class="literal-block">
lastal hugeDb0 queries.fasta &gt; myalns0.maf
lastal hugeDb1 queries.fasta &gt; myalns1.maf
lastal hugeDb2 queries.fasta &gt; myalns2.maf
</pre>
<p>The former method reads the queries in large batches, and aligns each
batch to one volume at a time.  If you run several processes in
parallel, they will not necessarily use the same volume at the same
time.</p>
<p>Therefore, with parallel processes, you should either ensure you have
enough memory to hold several volumes simultaneously, or run lastal on
one volume at a time.  An efficient scheme is to use a different
computer for each volume.</p>
</div>
<div class="section" id="lastal8">
<h2>lastal8</h2>
<p>lastal8 has identical usage to lastal, and is used with <a class="reference external" href="lastdb.html">lastdb8</a>.  lastal cannot read the output of lastdb8, and
lastal8 cannot read the output of lastdb.</p>
</div>
</div>
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</html>