<html><head></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; color: rgb(0, 0, 0); font-size: 14px; font-family: Calibri, sans-serif;"><div>All I did was supply the cDNA to est= and the scaffolds to genome=. All the other parameters I just used the default, then I ran MAKER. I am using version 2.30.</div><div><br></div><div>Thanks,</div><div>Carson</div><div><br></div><div><br></div><span id="OLK_SRC_BODY_SECTION"><div style="font-family:Calibri; font-size:11pt; text-align:left; color:black; BORDER-BOTTOM: medium none; BORDER-LEFT: medium none; PADDING-BOTTOM: 0in; PADDING-LEFT: 0in; PADDING-RIGHT: 0in; BORDER-TOP: #b5c4df 1pt solid; BORDER-RIGHT: medium none; PADDING-TOP: 3pt"><span style="font-weight:bold">From: </span> Xin Huang <<a href="mailto:xh33@georgetown.edu">xh33@georgetown.edu</a>><br><span style="font-weight:bold">Date: </span> Wednesday, November 13, 2013 at 8:45 PM<br><span style="font-weight:bold">To: </span> Carson Holt <<a href="mailto:carsonhh@gmail.com">carsonhh@gmail.com</a>><br><span style="font-weight:bold">Cc: </span> <<a href="mailto:maker-devel@yandell-lab.org">maker-devel@yandell-lab.org</a>><br><span style="font-weight:bold">Subject: </span> Re: [maker-devel] A suspected strange behavior of Maker...<br></div><div><br></div><div dir="ltr">Hi Carson,<div><br></div><div>Thanks a lot for doing the test for me! I didn't get any annotation in the GFF file generated by Maker even if I only used scaffold5347 as the genome and the face EST sequence as the est evidence. Is there something that I can modify to put the alignment into the GFF file? I used est2genome=1 option to get prediction from exonerate, but still couldn't get any prediction.</div><div><br></div><div>Thanks!</div><div><br></div><div>Xin</div></div><div class="gmail_extra"><br><br><div class="gmail_quote">On Wed, Nov 13, 2013 at 9:59 PM, Carson Holt <span dir="ltr"><<a href="mailto:carsonhh@gmail.com" target="_blank">carsonhh@gmail.com</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div style="font-size:14px;font-family:Calibri,sans-serif;word-wrap:break-word"><div>For scaffold5347 I get results from exonerate and blastn without any modification to default parameters. For scaffold3928 the alignment is very poor, and gets filtered out. I can force it to keep it but I have to allow for abnormally long introns of 65kb (introns that large are extremely rare in biology – as in if you took every gene from 100 organisms you might find a single gene among all of them with that long of an intron), and I have to allow for low end to end matching (less 30%). This alignment definitely should have been rejected.</div><div><br></div><div>Thanks,</div><div>Carson</div><div><br></div><div><br></div><span><div style="border-right:medium none;padding-right:0in;padding-left:0in;padding-top:3pt;text-align:left;font-size:11pt;border-bottom:medium none;font-family:Calibri;border-top:#b5c4df 1pt solid;padding-bottom:0in;border-left:medium none"><span style="font-weight:bold">From: </span> Xin Huang <<a href="mailto:xh33@georgetown.edu" target="_blank">xh33@georgetown.edu</a>><br><span style="font-weight:bold">Date: </span> Monday, November 11, 2013 at 8:52 AM<br><span style="font-weight:bold">To: </span> Carson Holt <<a href="mailto:carsonhh@gmail.com" target="_blank">carsonhh@gmail.com</a>><br><span style="font-weight:bold">Cc: </span> <<a href="mailto:maker-devel@yandell-lab.org" target="_blank">maker-devel@yandell-lab.org</a>><br><span style="font-weight:bold">Subject: </span> Re: [maker-devel] A suspected strange behavior of Maker...<br></div><div><div class="h5"><div><br></div><div dir="ltr">Hi Carson,<div><br></div><div>Thank you very much for getting back to me on this. Sorry I was too quick to jump to a suspicion! Thanks for pointing it out and please see attached the EST (face_cDNA.fa) and genome scaffold sequences (albo_scaf3928+5347_for_face_cDNA.fa) I used for Maker. We used blastn to identify these two scaffolds for this cDNA sequence, and exonerate result also showed that this cDNA can be aligned to the scaffolds.</div><div><br></div><div>Thanks!</div><div><br></div><div>Xin </div></div><div class="gmail_extra"><br><br><div class="gmail_quote">On Mon, Nov 11, 2013 at 12:10 AM, Carson Holt <span dir="ltr"><<a href="mailto:carsonhh@gmail.com" target="_blank">carsonhh@gmail.com</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div style="font-size:14px;font-family:Calibri,sans-serif;word-wrap:break-word"><div>Sorry for the slow reply, I was moving from Canada to the US, got sick, and my moving truck got delayed one week. So I fell behind on the maker list.</div><div><br></div><div>What you observe is correct behavior. The config is blasted against the database of ESTs/proteins. It really doesn't matter which way you blast, you pick the direction that is most efficient for the question you are asking, and you only have to adjust the Z value for database size if you want alignment scores to be reproducible both ways.</div><div><br></div><div>Not getting results means they were filtered out for some reason (you don't even have blastn results in your output). If you can send a test dataset I can take a look and tell you which filter.</div><div><br></div><div>Thanks,</div><div>Carson</div><div><br></div><div><br></div><span><div style="border-right:medium none;padding-right:0in;padding-left:0in;padding-top:3pt;text-align:left;font-size:11pt;border-bottom:medium none;font-family:Calibri;border-top:#b5c4df 1pt solid;padding-bottom:0in;border-left:medium none"><span style="font-weight:bold">From: </span> Xin Huang <<a href="mailto:xh33@georgetown.edu" target="_blank">xh33@georgetown.edu</a>><br><span style="font-weight:bold">Date: </span> Tuesday, November 5, 2013 2:40 PM<br><span style="font-weight:bold">To: </span> <<a href="mailto:maker-devel@yandell-lab.org" target="_blank">maker-devel@yandell-lab.org</a>><br><span style="font-weight:bold">Subject: </span> [maker-devel] A suspected strange behavior of Maker...<br></div><div><div><div><br></div><div dir="ltr">Hi,<div><br></div><div><span style="font-family: arial, sans-serif; font-size: 13px;">I found a very bizarre thing when I ran Maker several times. The blastn research seems to reverse query and subject the way it's specified in the maker_opts.ctl file. When I put the scaffold sequence after genome= and the cDNA sequence after est=, the blastn result actually used the scaffold as query and the cDNA as subject. I realized that this sounds unreal, so I reversed the places for the two and the blastn result is normal with the cDNA as query and the scaffold as subject, but at the same time, the GFF file is of the cDNA sequence. I tried a few times, and got the same result. I deleted Maker (2.28) and reinstalled the beta version (2.30), still getting the same result.</span><br></div><div><span style="font-family: arial, sans-serif; font-size: 13px;"><br></span></div><div><span style="font-family: arial, sans-serif; font-size: 13px;">My wild guess was that this might be the reason why the est2genome prediction using the EST sequence didn't go into the GFF3 file, as stated in a previous email to the Maker-devel. I couldn't figure out how to test this, though. I wonder if there's anything dramatically wrong that I've been doing to get such odd results... If you'd like me to send you any output files or control files, please let me know and I'll promptly pass those along.</span></div><div><span style="font-family: arial, sans-serif; font-size: 13px;"><br></span></div><div><span style="font-family: arial, sans-serif; font-size: 13px;">Thank you very much for considering my email.</span></div><div><span style="font-family: arial, sans-serif; font-size: 13px;"><br></span></div><div><span style="font-family: arial, sans-serif; font-size: 13px;">Sincerely,</span></div><div><span style="font-family: arial, sans-serif; font-size: 13px;"><br></span></div><div><span style="font-family: arial, sans-serif; font-size: 13px;">Xin Huang</span></div></div></div></div>
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