<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="">Hi Lahcen,<div class=""><br class=""></div><div class="">I put some answers below.<br class=""><div><blockquote type="cite" class=""><div class="">On Nov 15, 2017, at 11:32 AM, lahcen campbell <<a href="mailto:lahcencampbell@gmail.com" class="">lahcencampbell@gmail.com</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><div dir="ltr" class="">Hi Michael and Carson<div class=""><br class=""></div><div class="">Thank you both for your helpful input, I really appreciate it. <br class=""></div><div class=""><br class=""></div><div class="">See below for my comments...</div><div class=""><br class=""></div><div class="">Best</div><div class="">Lahcen</div><div class=""><br class=""></div><div class="gmail_extra"><br class=""><div class="gmail_quote">On Tue, Nov 14, 2017 at 5:04 PM, Michael Campbell <span dir="ltr" class=""><<a href="mailto:michael.s.campbell1@gmail.com" target="_blank" class="">michael.s.campbell1@gmail.com</a>></span> wrote:<br class=""><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div style="word-wrap:break-word" class="">Hi Lancen,<div class=""><br class=""></div><div class="">Thanks, the name has served me well for a number of years now :)</div></div></blockquote><div class=""><br class=""></div><div class="">Its a good name, I wouldn't change it haha :) </div><div class=""> </div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div style="word-wrap:break-word" class=""><div class=""><br class=""></div><div class="">So I started a run with your 11 scaffolds. I gave it the protein file that you sent and used all of repbase for masking. All of the scaffolds finished without error. I was hoping it would be something simple that just needed another set of eyes to see, looks like it's not the case for this one.</div><div class=""><br class=""></div><div class="">To further rule out a data issue I would try running it with the dpp test data that is bundled with MAKER to see if you can get the same error. This data set will run in about a minute. If you are on a cluster I would try running it with and without submitting it you the nodes and with and without mpi.</div><div class=""><br class=""></div><div class="">One thing that I have done in the past is to make a new directory and run maker there (this doesn't make a lot of sense but when the error doesn't make sense either it seems reasonable). </div></div></blockquote><div class=""><br class=""></div><div class=""><div class="">First off, I can report good news regards the 0 lengths contigs I was getting back. Carson, your thoughts on Bioperl conflict issues seemed to be the main issue. Out cluster software environment had gone through some changes of late, so working off the basis of that I was able to load the right bash config which resulted in no more 0 length contig errors. Huzzah !! </div></div><div class=""><font color="#38571a" class="">Great</font></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div style="word-wrap:break-word" class=""><div class=""><br class=""></div><div class="">As far as rerunning MAKER there are a couple of approaches. If you want it to stop complaining about trying to many times on failed contigs you can increase the number of tries in the opts file. The line looks like this:</div><div class=""><br class=""></div><div class="">tries=2 #number of times to try a contig if there is a failure for some reason</div><div class=""><br class=""></div><div class="">If you want to run it elsewhere, but you don't want to have to redo all of the repeat masking and blasting you can use the gff3 output from an earlier run. If you used gff3_merge after the first run finished you got a big gff3 file with all of the gene models and evidence. If you break up that file by the source column you can selectively pass the evidence back to MAKER. If you put all of the repeatmasker and repeatrunner entries into one file and pass it in on this line:</div></div></blockquote><div class=""><br class=""></div><div class=""><div class="">Can I ask, because I can't seem to find any concrete info on best practices for parsing MAKER gffs to partition the various source column fields as you described Michael. <br class=""></div><div class=""><br class=""></div><div class="">Is there a commonly used way to partition MAKER gffs based on source column? Or will I need to code it up, I ask because I feel this must have been needed before many times by other users. </div></div><div class=""> <font color="#38571a" class="">I've got a script that will do it if you want it. Since you don't need all of the entries grep is probably as easy as anyting. grep -P '\tsource\t'</font></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div style="word-wrap:break-word" class=""><div class=""><br class=""></div><div class="">rm_gff= #pre-identified repeat elements from an external GFF3 file</div></div></blockquote><div class=""><br class=""></div><div class="">I will remove links to fasta files for both 'rmlib=' and 'repeat_protein='</div>
<div class=""><font color="#38571a" class="">Yep</font></div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div style="word-wrap:break-word" class=""><div class=""><br class=""></div><div class="">you can turn off model_org= and repeat_protein=. This will speed up the next run a lot. Then you can pass in the protein2genome gff3 data on this line:</div><div class=""><br class=""></div><div class="">protein_gff= #aligned protein homology evidence from an external GFF3 file</div><div class=""><br class=""></div><div class="">Don't pass the blast gff3 data in. If you pass in gff3 data to maker is assumes that it is polished and will not make any effort to fix alignments. the protein2genome data is polished. est2genome is the equivalent for EST input.</div></div></blockquote><div class=""><br class=""></div><div class="">You say don't pass the blast as gff. As I pass in all other info via GFF3 and remove any evidence as fasta inputs... BLAST won't be called again right ? Ensuring the shortest possible rerun of MAKER to roll back to a uncorrupted state. </div><div class=""><font color="#38571a" class="">Right. blast will not be called as long as you remove or comment out the paths to the fastas in the est= and protein= lines.</font></div></div></div></div></div></blockquote><br class=""><blockquote type="cite" class=""><div class=""><div dir="ltr" class=""><div class="gmail_extra"><div class="gmail_quote"><div class=""><div class="">I noticed that the only unique source field types in my MAKER GFF are as follows: </div><div class=""><div class=""><i class=""><b class="">augustus_masked </b></i></div><div class=""><i class=""><b class="">blastx</b></i></div><div class=""><i class=""><b class="">maker</b></i></div><div class=""><i class=""><b class="">protein2genome</b></i></div><div class=""><i class=""><b class="">repeatmasker</b></i></div><div class=""><i class=""><b class="">repeatrunner</b></i></div></div><div class=""><font color="#38571a" class="">That look right for the run you described</font></div><div class="">I read on the dev group that passing est evidence as GFF won't actually call Exonerate, est2genome <span style="font-family:Arial,Helvetica,sans-serif;font-size:13px" class="">option just tells MAKER to try and turn polished EST alignments directly into genes.... so If I pass this info again as GFF it will simply use the same info as it did originally and not have to recompute anything ? </span></div><div class=""><span style="font-family:Arial,Helvetica,sans-serif;font-size:13px" class=""><br class=""></span></div><div class=""><span style="font-family:Arial,Helvetica,sans-serif;font-size:13px" class="">Based on the above fields contained in my MAKER gff, which of the following options should I select to re-annotate based on this older run ? I suspect all the options below in green should be set to 1, and the others in red set to 0. </span></div><div class=""><span style="font-family:Arial,Helvetica,sans-serif;font-size:13px" class=""><br class=""></span></div><div class=""><div class=""><b class="">#-----Re-annotation Using MAKER Derived GFF3</b></div><div class="">.....</div><div class=""><i style="background-color:rgb(147,196,125)" class="">est_pass=1 #use ESTs in maker_gff: 1 = yes, 0 = no</i></div><div class=""><i style="background-color:rgb(204,0,0)" class="">altest_pass=0 #use alternate organism ESTs in maker_gff: 1 = yes, 0 = no</i></div><div class=""><i style="background-color:rgb(147,196,125)" class="">protein_pass=1 #use protein alignments in maker_gff: 1 = yes, 0 = no</i></div><div class=""><i style="background-color:rgb(147,196,125)" class="">rm_pass=1 #use repeats in maker_gff: 1 = yes, 0 = no</i></div><div class=""><i style="background-color:rgb(147,196,125)" class="">model_pass=1 #use gene models in maker_gff: 1 = yes, 0 = no</i></div><div class=""><i style="background-color:rgb(147,196,125)" class="">pred_pass=1 #use ab-initio predictions in maker_gff: 1 = yes, 0 = no</i></div><div class=""><i style="background-color:rgb(204,0,0)" class="">other_pass=0 #passthrough anyything else in maker_gff: 1 = yes, 0 = no </i></div></div><div class=""><i class=""><br class=""></i></div></div></div></div></div></div></blockquote><font color="#38571a" class="">You don't need model_pass or pred_pass if you plan on running gene finders</font><br class=""><blockquote type="cite" class=""><div class=""><div dir="ltr" class=""><div class="gmail_extra"><div class="gmail_quote"><div class=""><div class="">I don't think I will pass back anything under augustus_masked as I didn't set that up correctly initially, instead passing in a precomputed augustus gff which Im told isn't the best way to run MAKER. So if I can get back to a state of not failing all contigs, I will run Augustus inside maker itself on the 2nd pass. Note though, I am aware of the order of things normally, but for this instance I will continue with what I have done with success previously. </div></div></div></div></div></div></blockquote><font color="#38571a" class="">Yeah, when I have issues with failing contigs I'll pull stuff out until it starts running without error, then I add things back until something breaks.</font></div><div><font color="#38571a" class=""><br class=""></font><blockquote type="cite" class=""><div class=""><div dir="ltr" class=""><div class="gmail_extra"><div class="gmail_quote"><div class=""><div class="">Lastly, as this next run will be updating based on previous generated MAKER gff data.... what states should <font class="">est2genome and protein2genome be ? 1 or 0 ? </font></div></div></div></div></div></div></blockquote><font color="#38571a" class="">0 those options are just for generating gene models directly from evidence when you don't have any gene finders trained. When you say updating do you mean reusing evidence from previous runs and generating new gene annotations or are you taking existing gene models and adding new evidence to see if they can be improved?</font><br class=""><blockquote type="cite" class=""><div class=""><div dir="ltr" class=""><div class="gmail_extra"><div class="gmail_quote"><div class=""><div class=""><font class=""><br class=""></font></div><div class=""><font class="">Apologies for the lengthy email reply Michael. Much appreciated again, thank you !! </font></div></div></div></div></div></div></blockquote><font color="#38571a" class="">No Worries, hope it helps.</font><br class=""><blockquote type="cite" class=""><div class=""><div dir="ltr" class=""><div class="gmail_extra"><div class="gmail_quote"><div class=""><div class=""><font class=""><br class=""></font></div><div class=""><font class="">L</font></div>
<div class=""><br class=""></div></div><div class=""> </div><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div style="word-wrap:break-word" class=""><div class=""></div><div class="">Clean_up is useful if you are running on a file system that limits the number of files that you can write. It removes all of the intermediate files used in the annotation. This takes away the advantage of rerunning in the same directory. clean_try deletes everything first, and starts again. clean_try is the one that deletes everything and pretends that the first run never happened. </div><div class=""><br class=""></div><div class="">I ccd the list on this response just Incas anyone else has any ideas or is facing the same error.</div><div class=""><br class=""></div><div class="">Let me know if any of this helps,</div><div class="">Mike</div><div class=""><div class="gmail-h5"><div class=""><br class=""><div class=""><blockquote type="cite" class=""><div class="">On Nov 14, 2017, at 10:48 AM, lahcen campbell <<a href="mailto:lahcencampbell@gmail.com" target="_blank" class="">lahcencampbell@gmail.com</a>> wrote:</div><br class="gmail-m_-244735716841605874Apple-interchange-newline"><div class=""><div dir="ltr" class="">Hi Michael <div class=""><br class=""></div><div class="">Nice name btw I have a Michael in my name too :) Lahcen Michael Campbell to be exact haha...anyway... thanks for the reply and offer to help. </div><div class=""><br class=""></div><div class="">I have attached the file in question below. Its so strange, I had to just leave it alone cause it was making me quite frustrated. Those bugs which there are now common sense solutions are the worst cause very easily you reach a wall. </div><div class=""><br class=""></div><div class="">Might it have anything at all to do with the Protein homology file I passed in ? Though, note.... the same protein files here have been used in another maker run without issue so I kind of ruled that out already.....but just spitballing at this stage. </div><div class=""><br class=""></div><div class=""><br class=""></div><div class="">Might I be so cheeky to ask you one more MAKER related question Michael... ? Feel free to ignore it I hate to push but im desperate to figure it out with little time to do so... <br class=""></div><div class=""><br class=""></div><div class="">I have an issue with a different MAKER analysis. Currently any new run I attempt on this datastore, which has one round successful with 25000 odd genes and double the transcripts. I attempted to run the second round with a SNAP trained hmm (first time passing in SNAP hmm following first round EST/Protein evidence). In this attempt, because we obtained so many genes I thought I would be more stringent by changing the AED to 0.7 from 1.0. Something I see now I didn't approach in the right way... too late now sadly.</div><div class=""><br class=""></div><div class="">MAKER finishes fine, but now it views all previous scaffolds as FAILED. Nothing seems to change this and now the datastore is for all intents and purposes locked in failed state. It keeps mentioning changes to the opts file which there were, and that the previous runs didn't finish so it must delete them. The results obtained from round 1 are still there though Im pretty sure of that, all blast files etc are still there and populated. </div><div class=""><br class=""></div><div class="">Can you tell me the main differences either clean_up or clean_try have and which will completely and irreversibly wipe the first run? Something I don't want to repeat, just allow me to progress to the next round. Im hesitant to run them, but I've backed up the datastore incase. My next attempt will be to pass the exact same maker_opts file from the round1 run, with the only change made to clean_try/clean_up....Is this approach misguided ? </div><div class=""><br class=""></div><div class="">Your help is very much appreciated Michael so thank you, </div><div class="">Best</div><div class="">L</div><div class=""><br class=""></div><div class=""><div class="gmail_chip gmail_drive_chip" style="width:396px;height:18px;max-height:18px;background-color:rgb(245,245,245);padding:5px;color:rgb(34,34,34);font-family:arial;font-style:normal;font-weight:bold;font-size:13px;border:1px solid rgb(221,221,221);line-height:1"><a href="https://drive.google.com/file/d/19ooxfIUGygyW9GBY8uBwCYwjAywRWiL_/view?usp=drive_web" style="display:inline-block;max-width:366px;overflow:hidden;text-overflow:ellipsis;white-space:nowrap;text-decoration:none;padding:1px 0px;border:none" target="_blank" class=""><img style="vertical-align: bottom; border: none;" src="https://ssl.gstatic.com/docs/doclist/images/icon_10_generic_list.png" class=""> <span dir="ltr" style="color:rgb(17,85,204);text-decoration:none;vertical-align:bottom" class="">Combined_Protein_homology.fa.<wbr class="">zip</span></a><img style="opacity: 0.55; float: right; display: none;" class=""></div><div class="gmail_chip gmail_drive_chip" style="width:396px;height:18px;max-height:18px;background-color:rgb(245,245,245);padding:5px;color:rgb(34,34,34);font-family:arial;font-style:normal;font-weight:bold;font-size:13px;border:1px solid rgb(221,221,221);line-height:1"><a href="https://drive.google.com/file/d/1Mwj6Jpf1U9xzQVgxVFqeYyQokFIrDFo5/view?usp=drive_web" style="display:inline-block;max-width:366px;overflow:hidden;text-overflow:ellipsis;white-space:nowrap;text-decoration:none;padding:1px 0px;border:none" target="_blank" class=""><img style="vertical-align: bottom; border: none;" src="https://ssl.gstatic.com/docs/doclist/images/icon_10_generic_list.png" class=""> <span dir="ltr" style="color:rgb(17,85,204);text-decoration:none;vertical-align:bottom" class="">SubsampledGenomeFile_n10_<wbr class="">11MB.fasta</span></a><img style="opacity: 0.55; float: right; display: none;" class=""></div><br class=""></div><div class=""><br class=""></div><div class=""><br class=""></div></div><div class="gmail_extra"><br class=""><div class="gmail_quote">On Tue, Nov 14, 2017 at 3:08 PM, Michael Campbell <span dir="ltr" class=""><<a href="mailto:michael.s.campbell1@gmail.com" target="_blank" class="">michael.s.campbell1@gmail.com</a><wbr class="">></span> wrote:<br class=""><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left:1px solid rgb(204,204,204);padding-left:1ex"><div style="word-wrap:break-word" class="">Hi Lahcen,<div class=""><br class=""></div><div class="">Nothing comes right to mind for what could be causing this error. If you want to compress your FASTA and send it to me I can try and recreate the error and try and debug it.</div><div class=""><br class=""></div><div class="">Thanks,</div><div class="">Mike<br class=""><div class=""><blockquote type="cite" class=""><div class=""><div class="gmail-m_-244735716841605874h5"><div class="">On Nov 14, 2017, at 7:15 AM, lahcen campbell <<a href="mailto:lahcencampbell@gmail.com" target="_blank" class="">lahcencampbell@gmail.com</a>> wrote:</div><br class="gmail-m_-244735716841605874m_-8577554359614042639Apple-interchange-newline"></div></div><div class=""><div class=""><div class="gmail-m_-244735716841605874h5"><div dir="ltr" class="">Hi MAKER community,<div class=""><br class=""></div><div class="">I was hoping someone could help me. I have a very unusual error with two different versions of maker I have tested so far. This error shouldn't be happening but it occurs time and again no matter what I try. I have tried using 2.31.6_mpich3_icc and 2.31_mpi<wbr class="">ch3</div><div class=""><br class=""></div><div class="">Note that version 2.31.6_mpich3_icc is one I have used countless times and produced final MAKER annotations without issue. So its not that this version has issues to date. </div><div class=""><br class=""></div><div class="">Basically, this is a brand new MAKER analysis, I am only trying to train SNAP in this first round. I am following the MakerTutorial as documented this time around and I can't get past the initial SNAP train stage. </div><div class=""><br class=""></div><div class="">I have a single genome file with, 10 Long scaffolds making up just under 11MB (subsampled from my original full length assembly) of sequence data in which to train SNAP. The fasta file is not corrupted, and has been generated in various ways in order to test formatting issues etc. </div><div class=""><br class=""></div><div class="">I have only edited the maker_opts file and changed:<br class=""></div><div class=""><br class=""></div><div class=""><b class="">genome=</b></div><div class=""><b class="">protein=</b></div><div class=""><b class="">protein2genome=1</b></div><div class=""><br class=""></div><div class="">But see attached my maker CTL files. </div><div class=""><br class=""></div><div class="">The error consistently returned to me:</div><div class=""><br class=""></div><div class=""><div class=""><b class=""><i class="">Skipping the contig because it is too short!!</i></b></div><div class=""><b class=""><i class="">SeqID: contig_WHATEVER</i></b></div><div class=""><b class=""><i class="">Length: 0</i></b></div></div><div class=""><br class=""></div><div class=""><i class="">The sequences are no where near too short. This was verified independently outside maker to be sure. </i></div><div class=""><i class=""><br class=""></i></div><div class=""><i class="">The headers are as follows:</i></div><div class=""><div class=""><br class=""></div><div class="">>tig00000458 len=2889428 reads=4143 covStat=1793.77 gappedBases=no class=contig suggestRepeat=no suggestCircular=no</div><div class="">>tig00000159 len=3515005 reads=5100 covStat=2143.94 gappedBases=no class=contig suggestRepeat=no suggestCircular=no</div><div class="">>tig00006117 len=1009519 reads=1168 covStat=804.93 gappedBases=no class=contig suggestRepeat=no suggestCircular=no</div><div class="">>tig00000419 len=2633986 reads=3938 covStat=1519.93 gappedBases=no class=contig suggestRepeat=no suggestCircular=no</div><div class="">>tig00027677 len=108573 reads=86 covStat=86.05 gappedBases=no class=contig suggestRepeat=no suggestCircular=no</div><div class="">>tig00021790 len=202251 reads=158 covStat=184.12 gappedBases=no class=contig suggestRepeat=no suggestCircular=no</div><div class="">>tig00316948 len=280333 reads=237 covStat=253.23 gappedBases=no class=contig suggestRepeat=no suggestCircular=no</div><div class="">>tig00019606 len=149709 reads=82 covStat=150.02 gappedBases=no class=contig suggestRepeat=no suggestCircular=no</div><div class="">>tig00023852 len=189461 reads=115 covStat=192.28 gappedBases=no class=contig suggestRepeat=no suggestCircular=no</div><div class="">>tig00316994 len=19742 reads=1 covStat=0.00 gappedBases=no class=contig suggestRepeat=no suggestCircular=no</div><div class=""><br class=""></div><div class="">I have just about given up, I have no idea why its happening it makes zero sense. </div><div class=""><br class=""></div><div class="">Any help or information as to why this might be happening would be amazing. </div><div class=""><br class=""></div><div class="">Thank you in advance. </div><div class="">Lahcen</div><div class=""><br class=""></div>-- <br class=""><div class="gmail-m_-244735716841605874m_-8577554359614042639gmail_signature"><div dir="ltr" class=""><div class=""><div dir="ltr" class=""><div class=""><div dir="ltr" class=""><div class=""><div dir="ltr" class="">==============================<wbr class="">============<br class="">> Dr. Lahcen Campbell <<div class="">> Contact: <a href="mailto:lahcencampbell@gmail.com" target="_blank" class="">lahcencampbell@gmail.com</a> <</div><div class="">> <a href="https://www.ebi.ac.uk/about/people/lahcen-campbell" target="_blank" class="">https://www.ebi.ac.uk/about/<wbr class="">people/lahcen-campbell</a> <</div><div class=""><div class=""><div class="">==============================<wbr class="">============</div></div></div></div></div></div></div></div></div></div></div>
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