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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>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>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>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>I’ll try tweaking the <code>$min_intron</code> parameter. Could this parameter be added to the <code>maker_opts.ctl</code> configuration file?</p>
<p>Thanks for your help, Carson. Cheers,<br>
Shaun</p>
<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_1437072884178863104" 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 10:55:32 , 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>
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).
<div class=""><br class=""></div>
<div class="">The minimum intron given to exonerate for polishing
is 20. It’s hard coded, and you would have to manually edit
it.</div>
<div class=""><br class=""></div>
<div class="">Line 1534 in maker/lib/GI.pm —>
my $min_intron = 20; </div>
<div class="">
<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 15, 2015, at 6:36 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="">I’m using<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="">protein</code><span class="Apple-converted-space"> </span>evidence and<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="">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?</p>
<p style="margin: 15px 0px;" class="">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 style="margin: 15px 0px;" class="">Thanks,<br style="-webkit-margin-before: 0px;" class="">
Shaun</p>
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<br class="">
<div id="bloop_sign_1437006582812233984" 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>
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