<html><head><meta http-equiv="Content-Type" content="text/html charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;" class="">Thanks for the patch. I plan on adding the min_intron option as well as a snoscan bug fix to the stable release of MAKER soon. No plans for a GitHub move at present.<div class=""><br class=""></div><div class="">—Carson</div><div class=""><br class=""><div class=""><br class=""><div><blockquote type="cite" class=""><div class="">On Jul 30, 2015, at 5:21 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="">Increasing<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="">$min_intron</code><span class="Apple-converted-space"> </span>to<span class="Apple-converted-space"> </span><code style="font-family: Menlo, Consolas, 'Liberation Mono', Courier, monospace; font-size: 10pt; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; background-color: rgb(248, 248, 248); color: inherit; border: 1px solid rgb(234, 234, 234); margin: 0px 2px; padding: 0px 5px; word-break: normal; word-wrap: normal;" class="">250</code><span class="Apple-converted-space"> </span>did the trick! Thanks for the tip. For this organellar genome, all the introns (at least that I’ve found) are group II self-splicing introns, so they have a much larger minimum size (about 990 bp) than introns in other genomes.<span class="Apple-converted-space"> </span><a href="https://en.wikipedia.org/wiki/Group_II_intron" style="color: rgb(65, 131, 196); background-color: inherit; text-decoration: none;" class="">https://en.wikipedia.org/wiki/Group_II_intron</a></p><p style="margin: 15px 0px;" class="">Increasing<span class="Apple-converted-space"> </span><code style="font-family: Menlo, Consolas, 'Liberation Mono', Courier, monospace; font-size: 10pt; border-top-left-radius: 3px; border-top-right-radius: 3px; border-bottom-right-radius: 3px; border-bottom-left-radius: 3px; background-color: rgb(248, 248, 248); color: inherit; border: 1px solid rgb(234, 234, 234); margin: 0px 2px; padding: 0px 5px; word-break: normal; word-wrap: normal; -webkit-margin-before: 0px;" class="">$min_intron</code><span class="Apple-converted-space"> </span>worked perfectly for all but one gene. Decreasing<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="">--codongapopen</code><span class="Apple-converted-space"> </span>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="">--codongapextend</code><span class="Apple-converted-space"> </span>rescued that one gene, but I’ll probably just leave the defaults and annotate that one gene by hand.</p><p style="margin: 15px 0px;" class="">Note that<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>had no effect on<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>without the following patch. Will there be a stable release soon that includes this patch and 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="">min_intron</code><span class="Apple-converted-space"> </span>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 style="margin: 15px 0px;" class="">Have you considered moving the MAKER development to GitHub?</p><p style="margin: 15px 0px;" class="">Thanks again. Cheers,<br style="-webkit-margin-before: 0px;" class="">Shaun</p><pre style="margin: 15px 0px; 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(204, 204, 204); overflow: auto; padding: 4px 8px; word-break: normal; word-wrap: normal;" class=""><code class="diff" 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: 0px; margin: 0px; padding: 0px; word-break: normal; word-wrap: normal; -webkit-margin-before: 0px;">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><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_1438297826599222016" 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 13:10:13 , 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;"><span style="margin-top: 0px; margin-bottom: 0px;" class=""><div class="" style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;"><div class=""></div><div class="">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 class=""><blockquote type="cite" class="" style="margin: 15px 0px;"><div class="" style="margin-top: 0px;">On Jul 16, 2015, at 1:11 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:</div><br class="Apple-interchange-newline"><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;">Hi, Carson.</p><p class="" style="margin: 15px 0px;">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 class="" style="margin: 15px 0px;">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 class="" style="margin: 15px 0px;">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 class="" style="margin: 15px 0px;">I’ll try tweaking the<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;">$min_intron</code><span class="Apple-converted-space"> </span>parameter. Could this parameter be added to the<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;">maker_opts.ctl</code><span class="Apple-converted-space"> </span>configuration file?</p><p class="" style="margin: 15px 0px;">Thanks for your help, Carson. Cheers,<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></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" class="" style="font-family: Helvetica, Arial; font-size: 13px; margin: 0px;"><br class=""></div><br class=""><div id="bloop_sign_1437072884178863104" class="bloop_sign"><div class="" style="font-family: helvetica, arial; font-size: 13px;">-- <br class=""><a href="http://sjackman.ca/" class="" style="color: rgb(65, 131, 196); background-color: inherit; text-decoration: none;">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" class="" style="color: rgb(65, 131, 196); background-color: inherit; text-decoration: none;">carsonhh@gmail.com</a>) wrote:</p><blockquote type="cite" class="clean_bq" style="margin: 15px 0px;"><div class="" style="margin-top: 0px; margin-bottom: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;"><div class=""></div><div class=""><span class="" style="margin-top: 0px; margin-bottom: 0px;">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 class="" style="margin-top: 0px; margin-bottom: 0px;"><br class=""></span></div><div class=""><span class="" style="margin-top: 0px; margin-bottom: 0px;">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 class="" style="margin-top: 0px; margin-bottom: 0px;"><br class=""></span></div><div class=""><span class="" style="margin-top: 0px; margin-bottom: 0px;">Line 1534 in maker/lib/GI.pm —> my $min_intron = 20; </span></div><div class=""><div class=""><span class="" style="margin-top: 0px; margin-bottom: 0px;"><br class=""></span></div><div class=""><span class="" style="margin-top: 0px; margin-bottom: 0px;">—Carson</span></div><div class=""><span class="" style="margin-top: 0px; margin-bottom: 0px;"><br class=""></span></div><div class=""><span class="" style="margin-top: 0px; margin-bottom: 0px;"><br class=""></span><div class=""><blockquote type="cite" class="" style="margin: 15px 0px;"><div class="" style="margin-top: 0px;"><span class="" style="margin-top: 0px; margin-bottom: 0px;">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 class="" style="margin-top: 0px; margin-bottom: 0px;"><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 class="" style="-webkit-margin-before: 0px; margin-top: 0px; margin-bottom: 0px;">Hi, Carson.</span></p><p class="" style="margin: 15px 0px;"><span class="" style="-webkit-margin-before: 0px; margin-top: 0px; margin-bottom: 0px;">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></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" class="" style="font-family: Helvetica, Arial; font-size: 13px; margin: 0px;"><br class=""></div><br class=""><div id="bloop_sign_1437006582812233984" class="bloop_sign"><div class="" style="font-family: helvetica, arial; font-size: 13px;">-- <br class=""><a href="http://sjackman.ca/" class="" style="color: rgb(65, 131, 196); 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