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.error { color:#AA0000; }</style></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;"><div class="bloop_markdown"><p>Hi, Carson.</p>
<p>Increasing <code>$min_intron</code> to <code>250</code> did the trick! Thanks for the tip. For this organellar genome, all the introns (at least that I’ve found) are group II self-splicing introns, so they have a much larger minimum size (about 990 bp) than introns in other genomes. <a href="https://en.wikipedia.org/wiki/Group_II_intron">https://en.wikipedia.org/wiki/Group_II_intron</a></p>
<p>Increasing <code>$min_intron</code> worked perfectly for all but one gene. Decreasing <code>--codongapopen</code> and <code>--codongapextend</code> rescued that one gene, but I’ll probably just leave the defaults and annotate that one gene by hand.</p>
<p>Note that <code>$min_intron</code> had no effect on <code>protein2genome</code> without the following patch. Will there be a stable release soon that includes this patch and the <code>min_intron</code> option? I’m preparing a manuscript for submission, and I’d love to be able to refer to a stable version of MAKER in the manuscript that includes this feature.</p>
<p>Have you considered moving the MAKER development to GitHub?</p>
<p>Thanks again. Cheers,<br>
Shaun</p>
<pre><code class="diff">diff --git a/protein.pm.orig b/protein.pm
--- a/protein.pm.orig
+++ b/protein.pm
@@ -94,11 +94,11 @@ sub runExonerate {
my $command = "$exe -q $q_file -t $t_file -Q protein -T dna ";
$command .= "-m protein2genome --softmasktarget ";
+ $command .= " --minintron $min_intron --maxintron $max_intron --showcigar";
$command .= " --percent $percent";
if ($matrix) {
$command .= " --proteinsubmat $matrix";
}
- $command .= " --showcigar ";
$command .= " > $o_file";
my $w = new Widget::exonerate::protein2genome();
</code></pre>
<p></p></div><div class="bloop_original_html"><style>body{font-family:Helvetica,Arial;font-size:13px}</style><div id="bloop_customfont" style="font-family:Helvetica,Arial;font-size:13px; color: rgba(0,0,0,1.0); margin: 0px; line-height: auto;"><br></div> <br> <div id="bloop_sign_1438297826599222016" class="bloop_sign"><div style="font-family:helvetica,arial;font-size:13px">-- <br><a href="http://sjackman.ca/">http://sjackman.ca/</a></div></div> <br><p class="airmail_on" style="color:#000;">On 2015-July-16 at 13:10:13 , Carson Holt (<a href="mailto:carsonhh@gmail.com">carsonhh@gmail.com</a>) wrote:</p> <blockquote type="cite" class="clean_bq"><span><div style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class=""><div></div><div>
<title></title>
I can add it to the development version.
<div class=""><br class=""></div>
<div class="">—Carson</div>
<div class=""><br class=""></div>
<div class=""><br class="">
<div>
<blockquote type="cite" class="">
<div class="">On Jul 16, 2015, at 1:11 PM, Shaun Jackman
<<a href="mailto:sjackman@gmail.com" class="">sjackman@gmail.com</a>> wrote:</div>
<br class="Apple-interchange-newline">
<div class="">
<div class="bloop_markdown" style="font-family: Helvetica, Arial; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: rgb(254, 254, 254);">
<p style="margin: 15px 0px; -webkit-margin-before: 0px;" class="">
Hi, Carson.</p>
<p style="margin: 15px 0px;" class="">One of the ten questionable
introns has a canonical GT-AG splice site and is 33 bp. The splice
sites are GA-AG, GG-GG, GC-AG, GT-CG, GA-AT, GG-AA, GG-AG, AT-TT,
GG-AT and GT-AG. The intron sizes are 33, 111, 84, 30, 219, 186,
51, 30, 45 and 33.</p>
<p style="margin: 15px 0px;" class="">I was wrong about there being
stop codons in the questionable introns. All ten are completely
free of stop codons. Sorry for the confusion. I had extracted just
the intron sequence and translated the first frame, but the intron
was not aligned to a 3-nucleotide boundary.</p>
<p style="margin: 15px 0px;" class="">I am convinced that these
short introns are in fact genomic insertions and not introns. The
root cause may be incorrectly annotated introns in the protein
evidence, as you suggest.</p>
<p style="margin: 15px 0px;" class="">I’ll try tweaking
the<span class="Apple-converted-space"> </span><code style="font-family: Menlo, Consolas, 'Liberation Mono', Courier, monospace; font-size: 10pt; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; background-color: rgb(248, 248, 248); color: inherit; border: 1px solid rgb(234, 234, 234); margin: 0px 2px; padding: 0px 5px; word-break: normal; word-wrap: normal; -webkit-margin-before: 0px;" class="">$min_intron</code><span class="Apple-converted-space"> </span>parameter. Could this
parameter be added to the<span class="Apple-converted-space"> </span><code style="font-family: Menlo, Consolas, 'Liberation Mono', Courier, monospace; font-size: 10pt; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; background-color: rgb(248, 248, 248); color: inherit; border: 1px solid rgb(234, 234, 234); margin: 0px 2px; padding: 0px 5px; word-break: normal; word-wrap: normal;" class="">maker_opts.ctl</code><span class="Apple-converted-space"> </span>configuration file?</p>
<p style="margin: 15px 0px;" class="">Thanks for your help, Carson.
Cheers,<br style="-webkit-margin-before: 0px;" class="">
Shaun</p>
<div style="margin: 15px 0px;" class=""><br class="webkit-block-placeholder"></div>
</div>
<div class="bloop_original_html" style="font-family: Helvetica, Arial; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: rgb(254, 254, 254);">
<div id="bloop_customfont" style="font-family: Helvetica, Arial; font-size: 13px; margin: 0px;" class=""><br class=""></div>
<br class="">
<div id="bloop_sign_1437072884178863104" class="bloop_sign">
<div style="font-family: helvetica, arial; font-size: 13px;" class="">-- <br class="">
<a href="http://sjackman.ca/" style="color: rgb(65, 131, 196); background-color: inherit; text-decoration: none;" class="">http://sjackman.ca/</a></div>
</div>
<br class="">
<p class="airmail_on" style="margin: 15px 0px;">On 2015-July-16 at
10:55:32 , Carson Holt (<a href="mailto:carsonhh@gmail.com" style="color: rgb(65, 131, 196); background-color: inherit; text-decoration: none;" class="">carsonhh@gmail.com</a>) wrote:</p>
<blockquote type="cite" class="clean_bq" style="margin: 15px 0px;">
<div class="" style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;">
<div class=""></div>
<div class=""><span style="margin-top: 0px; margin-bottom: 0px;" class="">Look at the region. If it’s being suggested by the
polished alignment, I somewhat doubt it’s just an insertion because
the polished alignment will have valid splice sites. It could
be an insertion, but one that perfectly maps around canonical
splice sites would be quite the coincidence (because exonerate
shouldn't make big gaps to force the alignment). However if it
looks more like a forced mapping around non-canonical spice sites
(which shouldn’t actually produce protein2genome results) then I
might support the idea that it’s an insertion. A 250bp intron
or even a 100 bp doesn’t really seem that short to me. The lower
range seen in fungi for example (which have very short introns) can
get close to about 20bp. I guess it’s possible that the
protein evidence you are using contains an intron that isn’t really
there, that results in an intron in your job because of protein
conservation (i.e. conserved codons contain the falsely used splice
site).</span>
<div class=""><span style="margin-top: 0px; margin-bottom: 0px;" class=""><br class=""></span></div>
<div class=""><span style="margin-top: 0px; margin-bottom: 0px;" class="">The minimum intron given to exonerate for polishing is 20.
It’s hard coded, and you would have to manually edit
it.</span></div>
<div class=""><span style="margin-top: 0px; margin-bottom: 0px;" class=""><br class=""></span></div>
<div class=""><span style="margin-top: 0px; margin-bottom: 0px;" class="">Line 1534 in maker/lib/GI.pm —>
my $min_intron = 20; </span></div>
<div class="">
<div class=""><span style="margin-top: 0px; margin-bottom: 0px;" class=""><br class=""></span></div>
<div class=""><span style="margin-top: 0px; margin-bottom: 0px;" class="">—Carson</span></div>
<div class=""><span style="margin-top: 0px; margin-bottom: 0px;" class=""><br class=""></span></div>
<div class=""><span style="margin-top: 0px; margin-bottom: 0px;" class=""><br class=""></span>
<div class="">
<blockquote type="cite" class="" style="margin: 15px 0px;">
<div class="" style="margin-top: 0px;"><span style="margin-top: 0px; margin-bottom: 0px;" class="">On Jul 15, 2015, at
6:36 PM, Shaun Jackman <<a href="mailto:sjackman@gmail.com" class="" style="color: rgb(65, 131, 196); background-color: inherit; text-decoration: none;">sjackman@gmail.com</a>>
wrote:</span></div>
<span style="margin-top: 0px; margin-bottom: 0px;" class=""><br class="Apple-interchange-newline"></span>
<div class="" style="margin-bottom: 0px;">
<div class="bloop_markdown" style="font-family: Helvetica, Arial; font-size: 13px; font-style: normal; font-variant: normal; font-weight: normal; letter-spacing: normal; line-height: normal; orphans: auto; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: auto; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: rgb(254, 254, 254);">
<p class="" style="margin: 15px 0px; -webkit-margin-before: 0px;">
<span style="margin-top: 0px; margin-bottom: 0px;" class="">Hi,
Carson.</span></p>
<p class="" style="margin: 15px 0px;"><span style="margin-top: 0px; margin-bottom: 0px;" class="">I’m
using<span class="Apple-converted-space"> </span><code class="" style="font-family: Menlo, Consolas, 'Liberation Mono', Courier, monospace; font-size: 10pt; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; background-color: rgb(248, 248, 248); color: inherit; border: 1px solid rgb(234, 234, 234); margin: 0px 2px; padding: 0px 5px; word-break: normal; word-wrap: normal; -webkit-margin-before: 0px;">protein</code><span class="Apple-converted-space"> </span>evidence
and<span class="Apple-converted-space"> </span><code class="" style="font-family: Menlo, Consolas, 'Liberation Mono', Courier, monospace; font-size: 10pt; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; background-color: rgb(248, 248, 248); color: inherit; border: 1px solid rgb(234, 234, 234); margin: 0px 2px; padding: 0px 5px; word-break: normal; word-wrap: normal;">protein2genome</code><span class="Apple-converted-space"> </span>alone
without<span class="Apple-converted-space"> </span><em class="">ab initio</em><span class="Apple-converted-space"> </span>gene finders to annotate an
organellar genome. MAKER annotates 16 introns. 6 introns look real
(according to RNAweasel) and are all larger than 900 bp. The other
10 introns are all shorter than 250 bp and multiples of 3 bp. These
short introns look like genomic insertions rather than introns to
me. Is there a way to specify a minimum intron size to
MAKER?</span></p>
<p class="" style="margin: 15px 0px;">6 of these short introns do
not contain a stop codon, and 4 do contain a stop codon. I suppose
these 4 are pseudogenes.</p>
<p class="" style="margin: 15px 0px;">Thanks,<br class="" style="-webkit-margin-before: 0px;">
Shaun</p>
<div class="" style="margin: 15px 0px;"><br class="webkit-block-placeholder" style="-webkit-margin-before: 0px;"></div>
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http://sjackman.ca/</a></div>
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