<html><head></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space; font-family: Calibri, sans-serif; "><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125); "><span>1.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; "> </span></span></span><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">To run maker, we use transcripts produced by tophat+cufflink approach instead of de novo trinity. Will it avoid the possible merging of RNA-Seq reads? </span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><br></span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">No. Trinity would probably be a better approach to avoid merging.</span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><br></span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><br></span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125); "><span>2.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; "> </span></span></span><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">If my understanding is correct, the “correct_est_fusion” parameter needs to be turned off when we don’t ask Maker/prediction algorithms to predict UTRs? Also, it makes me wonder, in such case, when Maker turn off UTRs, but our RNA-Seq data has got long UTRs, will these UTRs been added into the maker gene model? </span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><br></span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">MAKER will always try to add UTR if the EST evidence suggests it. Technically it's a little bit more than that, it can also add missing exons and extend CDS. The correct_est_fusion, just causes it to clip really long UTR if it looks like it was added due to merged evidence, and is probably not really a contiguous part of the gene. The long UTRs that can result from mRNA-seq are often false. You are basically expending the UTR by assembling into exons from the neighboring gene. This is especially common in organisms like fungi where UTR of neighboring genes often overlap, and mRNA-seq assemblies falsely make it look like one transcript encompasses 1, 2 , or more genes loci (you loose the true UTR boundaries).</span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><br></span></span></div><div><font class="Apple-style-span" color="#1f497d"><span class="Apple-style-span" style="font-size: 15px;">--Carson</span></font></div><div><font class="Apple-style-span" color="#1f497d"><span class="Apple-style-span" style="font-size: 15px;"><br></span></font></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><br></span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><br></span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><span class="Apple-style-span" style="font-family: 'Times New Roman', serif; font-size: 16px; "><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><br></span></span></div><div style="color: rgb(0, 0, 0); font-size: 14px; "><br></div><span id="OLK_SRC_BODY_SECTION" style="color: rgb(0, 0, 0); font-size: 14px; "><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> <<a href="mailto:Sean.Li@csiro.au">Sean.Li@csiro.au</a>><br><span style="font-weight:bold">Date: </span> Tuesday, 21 May, 2013 9:23 PM<br><span style="font-weight:bold">To: </span> Carson Holt <<a href="mailto:carsonhh@gmail.com">carsonhh@gmail.com</a>>, Barry Moore <<a href="mailto:barry.moore@genetics.utah.edu">barry.moore@genetics.utah.edu</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] Fused gene problem, improvement in the Maker 2.27?<br></div><div><br></div><div xmlns:v="urn:schemas-microsoft-com:vml" xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns:m="http://schemas.microsoft.com/office/2004/12/omml" xmlns="http://www.w3.org/TR/REC-html40"><meta http-equiv="Content-Type" content="text/html; charset=us-ascii"><meta name="Generator" content="Microsoft Word 12 (filtered medium)"><base href="x-msg://337/"><style><!--
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</o:shapelayout></xml><![endif]--><div lang="EN-AU" link="blue" vlink="purple"><div class="WordSection1"><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">Thanks Barry and Carson for your detailed explanation. Now I have a better understand of “pred_flank”.
<o:p></o:p></span></p><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><o:p> </o:p></span></p><p class="MsoListParagraph" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><span style="mso-list:Ignore">1.<span style="font:7.0pt "Times New Roman"">
</span></span></span><!--[endif]--><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">To run maker, we use transcripts produced by tophat+cufflink approach instead of de novo trinity. Will it avoid the possible merging of RNA-Seq reads?
<o:p></o:p></span></p><p class="MsoListParagraph" style="text-indent:-18.0pt;mso-list:l0 level1 lfo1"><!--[if !supportLists]--><span style="font-size:11.0pt;font-family:"Calibri","sans-serif";color:#1F497D"><span style="mso-list:Ignore">2.<span style="font:7.0pt "Times New Roman"">
</span></span></span><!--[endif]--><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">If my understanding is correct, the “correct_est_fusion” parameter needs to be turned off when we don’t ask Maker/prediction algorithms to predict
UTRs? Also, it makes me wonder, in such case, when Maker turn off UTRs, but our RNA-Seq data has got long UTRs, will these UTRs been added into the maker gene model? <o:p></o:p></span></p><p class="MsoListParagraph"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><o:p> </o:p></span></p><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">Regards,<o:p></o:p></span></p><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">Sean<o:p></o:p></span></p><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "><o:p> </o:p></span></p><div><div style="border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0cm 0cm 0cm"><p class="MsoNormal"><b><span lang="EN-US" style="font-size: 10pt; font-family: Tahoma, sans-serif; ">From:</span></b><span lang="EN-US" style="font-size: 10pt; font-family: Tahoma, sans-serif; "> Carson Holt [<a href="mailto:carsonhh@gmail.com">mailto:carsonhh@gmail.com</a>]
<br><b>Sent:</b> Wednesday, 22 May 2013 10:59 AM<br><b>To:</b> Barry Moore; Li, Sean (CMIS, Acton)<br><b>Cc:</b> <a href="mailto:maker-devel@yandell-lab.org">maker-devel@yandell-lab.org</a><br><b>Subject:</b> Re: [maker-devel] Fused gene problem, improvement in the Maker 2.27?<o:p></o:p></span></p></div></div><p class="MsoNormal"><o:p> </o:p></p><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; ">Yes. Barry gave a good overview. The correct_est_fusion option basically clips UTR when there are two neighboring genes that only overlap in the UTR (so you
still get both gene models). Since the primary effect of falsely merged mRNA-seq is overly long UTR this tends to fix many cases. Of course avoiding merging the mRNA-seq reads in the first place also works. So using Trinity's extra options to control that
together with the correct_est_option option in MAKER is probably the way to go.<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; ">I think you can lower pred_flank to 100, but below that you might start to get weird behavior from the gene predictors (they need some upstream and downstream
sequence or the HMMs don't work well).<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; ">Thanks,<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; ">Carson<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div><div style="border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0cm 0cm 0cm"><p class="MsoNormal"><b><span style="font-size: 11pt; color: black; font-family: Calibri, sans-serif; ">From:
</span></b><span style="font-size: 11pt; color: black; font-family: Calibri, sans-serif; ">Barry Moore <<a href="mailto:barry.moore@genetics.utah.edu">barry.moore@genetics.utah.edu</a>><br><b>Date: </b>Tuesday, 21 May, 2013 7:54 PM<br><b>To: </b><<a href="mailto:Sean.Li@csiro.au">Sean.Li@csiro.au</a>><br><b>Cc: </b><<a href="mailto:maker-devel@yandell-lab.org">maker-devel@yandell-lab.org</a>><br><b>Subject: </b>Re: [maker-devel] Fused gene problem, improvement in the Maker 2.27?<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div><div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; ">Hi Sean,<o:p></o:p></span></p><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; ">I think you want to be careful with dropping the pred_flank parameter too low. This controls how much flanking sequence (for a given cluster of evidence) MAKER
will pass to the gene predictor. Some (maybe all?) of the gene predictors have an initial state in their HMM for intergenic sequence and if you do not have some intergenic sequence for them to consider first they can't transition to their next state. The
correct_est_fusion option can help (at the cost of losing some UTR annotations) - Carson will likely give you a better description of the intricacies of the correct_est_fusion.<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; ">Don't know how you are assembling your RNASeq, but there is an option in Trinity - I forget the name - that will instruct Trinity to be more restrictive in merging
neighboring clusters of reads into a longer transcript and this can help as well.<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; ">B<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div><div><div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; ">On May 21, 2013, at 1:36 AM, <<a href="mailto:Sean.Li@csiro.au">Sean.Li@csiro.au</a>><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "> wrote:<o:p></o:p></span></p></div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><br><br><o:p></o:p></span></p><div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">Hi Carson,</span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "> </span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">We are currently working on the annotation of Helicoverpa genome project. Maker has been chosen as the preliminary tool for the task. By checking the annotation
results by using maker 2.10, we saw some loci have the fusion problem: two separate neighbour genes are likely to be fused together and regarded as a single candidate output by maker. If we go further by looking at the outputs from each individual de novo
algorithm, e.g. augustus or snap, the prediction was correct. We are also using RNA-Seq assembly from cufflinks and some protein evidence data from closely related insects. </span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "> </span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">We noticed that the parameters “pred_flank” in maker v2.10 and “correct_est_fusion” in maker v2.27 might be useful for maker to decide when to merge models
or not. If possible, can you please explain what these two parameters can do with the predicted genes, RNA-Seq and protein evidence?</span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "> </span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">Also, our current plan is to install maker 2.27, train the algorithms to predict UTRs, enlarge the protein evidence datasets and input our previous annotations
as model_gff. We are facing with an critical question: in which way we could effectively improve the gene fusing problem? 1) setting the pred_flank lower than 100? 2) turn the correct_est_fusion on? 3) anything else? </span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "> </span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">Thank you.</span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "> </span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">With best regards,</span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="color: rgb(31, 73, 125); font-family: Calibri, sans-serif; ">Xi (Sean) Li, Ph. D.<br><br>
Bioinformatics Analyst, Bioinformatics Core,<br>
CSIRO Mathematics, Informatics and Statistics<br>
Phone:<span class="apple-converted-space"> </span><a href="tel:%2B61%202%206216%207138" target="_blank">+61 2 6216 7138</a><br>
Address: GPO Box 664, Canberra, ACT 2601</span><span style="color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "> </span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "> </span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "> </span><span style="font-size:10.5pt;font-family:Consolas;color:black"><o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size:10.5pt;font-family:Consolas;color:black"> <o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 11pt; color: rgb(31, 73, 125); font-family: Calibri, sans-serif; "> </span><span style="color:black"><o:p></o:p></span></p></div><p class="MsoNormal"><span style="font-size: 13.5pt; color: black; font-family: Helvetica, sans-serif; ">_______________________________________________<br>
maker-devel mailing list<br><a href="mailto:maker-devel@box290.bluehost.com">maker-devel@box290.bluehost.com</a><br><a href="http://box290.bluehost.com/mailman/listinfo/maker-devel_yandell-lab.org">http://box290.bluehost.com/mailman/listinfo/maker-devel_yandell-lab.org</a><o:p></o:p></span></p></div></div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p><div><div><div><p class="MsoNormal"><span style="font-size: 9pt; color: black; font-family: Arial, sans-serif; ">Barry Moore<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 9pt; color: black; font-family: Arial, sans-serif; ">Research Scientist<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 9pt; color: black; font-family: Arial, sans-serif; ">Dept. of Human Genetics<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 9pt; color: black; font-family: Arial, sans-serif; ">University of Utah<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 9pt; color: black; font-family: Arial, sans-serif; ">Salt Lake City, UT 84112<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 9pt; color: black; font-family: Arial, sans-serif; ">--------------------------------------------<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 9pt; color: black; font-family: Arial, sans-serif; ">(801) 585-3543<o:p></o:p></span></p></div><div><p class="MsoNormal"><span style="font-size: 9pt; color: black; font-family: Arial, sans-serif; "><o:p> </o:p></span></p></div></div><div><p class="MsoNormal"><span style="font-size: 13.5pt; color: black; font-family: Helvetica, sans-serif; "><o:p> </o:p></span></p></div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; "><o:p> </o:p></span></p></div></div></div><p class="MsoNormal"><span style="font-size: 10.5pt; color: black; font-family: Calibri, sans-serif; ">_______________________________________________ maker-devel mailing list
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