<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 Pei-Ying,<div class=""><br class=""></div><div class="">The time it takes to run MAKER is a hard to guess because it is dependent on the size of the genome and the amount of evidence you give it. However, There may be more going on. Can you tell if MAKER is using all of the cores that you gave it? </div><div class=""><br class=""></div><div class="">For training augustus, there are several options. Using the CEGMA output is a common method. Given that your genome is a 4G plant genome I don’t think GeneMark will perform well. If you used the step you mentioned below but left GeneMark out you may get a better training than you would with CEGMA output alone.</div><div class=""><br class=""></div><div class="">I’ve ccd Carson Holt, he has much more experience with the MPI aspects of MAKER and may have some additional insights. I’m also ccing the devlist. There may be others in the community that can comment on the run times.</div><div class=""><br class=""></div><div class="">Thanks,</div><div class="">Mike<br class=""><div><blockquote type="cite" class=""><div class="">On May 17, 2016, at 10:10 PM, Pei-Ying Huang <<a href="mailto:themis.ray@gmail.com" class="">themis.ray@gmail.com</a>> wrote:</div><br class="Apple-interchange-newline"><div class=""><div dir="ltr" class="">Hi mike,<div class=""><br class=""></div><div class="">My plant genome is about 4Gb, 93789 scaffolds. When I run maker using MPI on a server with 64 cores, only 1% of genome is annotated.</div><div class="">Is it the normal condition? Since I read a post said that it takes about 6 days on 16 processor to finish one round on a ~150,000 scaffold ~2Gb vertebrate genome with protein evidence.</div><div class="">Then based on the post, I expect I get the result no more than two weeks. However, it seems it will take me more than three months.</div><div class=""><br class=""></div><div class="">Also I want to get a training set parameter by augustus, now I use CEGMA to produce a .gff file, then convert it to augustus.gff by cegma2gff.</div><div class="">Then autotrain with augustus, here is my command</div><div class="">autoAugTrain.pl --genome=GULI.genome.removeAllN.fa --trainingset=augustus.gff --species=A_autoAugTrain_1 &> log </div><div class=""><br class=""></div><div class=""><div class=""><br class=""></div><div class=""><div class="">But I saw one's method below, so I wonder if I am doing wrong?</div><div class=""><br class=""></div><div class="">"We get the genome.gff3 training set from the output of a first-pass run of MAKER using: </div><div class="">1. EST data</div><div class="">2. Proteins from related species </div><div class="">3. a SNAP model trained using CEGMA </div><div class="">4. a GeneMark model (obtained by running GeneMark.ES on the draft genome) </div><div class="">5. Running maker2zff on the output of MAKER, and converting that to GFF3</div><div class="">Once done, we run MAKER a second time using the Augustus model and more stringent settings."<br class=""></div><div class=""><br class=""></div><div class="">Thank you.</div></div></div><div class="">Pei-Ying</div><div class=""><br class=""></div><div class=""> <br class=""></div><div class=""><br class=""></div><div class=""><br class=""></div></div><div class="gmail_extra"><br class=""><div class="gmail_quote">2016-05-18 9:16 GMT+08:00 Michael Campbell <span dir="ltr" class=""><<a href="mailto:michael.s.campbell1@gmail.com" target="_blank" class="">michael.s.campbell1@gmail.com</a>></span>:<br class=""><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div style="word-wrap:break-word" class="">Hi Pei-Ying,<div class=""><br class=""></div><div class="">One of the first places to start with RNA-seq quality control is using a tool called fastqc it will produce a number of graphics that can help identify problematic files. There are a number of tools for quality trimming reads, timmomatic and fastx tools are popular ones. </div><div class=""><br class=""></div><div class="">I would only redo the sequencing if you are convinced that the original sequencing is bad.</div><div class=""><br class=""></div><div class="">Mike</div><div class=""><br class=""></div><div class=""><br class=""><div class=""><blockquote type="cite" class=""><div class="">On May 16, 2016, at 8:42 PM, Pei-Ying Huang <<a href="mailto:themis.ray@gmail.com" target="_blank" class="">themis.ray@gmail.com</a>> wrote:</div><br class=""><div class=""><div dir="ltr" class="">Hi mike,<div class=""><br class=""></div><div class=""><span style="font-size:14px" class="">As you said the reason I only get one gene with the transcript evidence is independent of MAKER and could be RNA-seq data quality or the expression profiles of the tissues used for mRNA-seq.</span></div><div class=""><span style="font-size:14px" class=""><br class=""></span></div><div class=""><span style="font-size:14px" class="">If the problem is due to RNA-seq data quality, how could I identify the </span><span style="font-size:14px" class="">RNA-seq data with bad quality and </span><span style="font-size:14px" class="">trim them out?</span></div><div class=""><span style="font-size:14px" class="">If the problem is due to </span><span style="font-size:14px" class="">expression profiles of the tissues used for mRNA-seq, should we try to extract RNA from the plant again and redo the sequencing?</span></div><div class=""><span style="font-size:14px" class="">Thank you.</span></div><div class=""><span style="font-size:14px" class=""><br class=""></span></div><div class=""><span style="font-size:14px" class="">Pei-Ying</span></div></div><div class="gmail_extra"><br class=""><div class="gmail_quote">2016-05-09 22:18 GMT+08:00 Michael Campbell <span dir="ltr" class=""><<a href="mailto:michael.s.campbell1@gmail.com" target="_blank" class="">michael.s.campbell1@gmail.com</a>></span>:<br class=""><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div style="word-wrap:break-word" class="">I did finish running the test I planned. What I noticed is that there is protein evidence for about 1,000 genes on that scaffold and transcript evidence for only one gene. The reason you only get one gene with the transcript evidence is independent of MAKER and could be RNA-seq data quality or the expression profiles of the tissues used for mRNA-seq. <div class=""><br class=""></div><div class="">What you described is what I would do. Followed by training augustus. Unless est2genome=1 and prtein2genome=0 doesn’t generate enough gene models to train the gene finders. Then I would set est2genome=1 and protein2genome=1 for the first round instead.</div><div class=""> </div><div class="">Thanks,<br class=""><div class="">Mike<br class=""><div class=""><blockquote type="cite" class=""><div class="">On May 8, 2016, at 10:08 AM, Pei-Ying Huang <<a href="mailto:themis.ray@gmail.com" target="_blank" class="">themis.ray@gmail.com</a>> wrote:</div><br class=""><div class=""><div dir="ltr" class="">Have you done all of the test?<div class="">What would you suggest me to run my data?</div><div class=""><br class=""></div><div class="">To get ab initio model by setting the est2genome =1 and protein2genome = 0,</div><div class="">then training with sanp model with est2genome = 0 and protein2genome = 0,</div><div class="">training second snap model with est2genome = 0 and protein2genome = 0.</div><div class=""><br class=""></div><div class="">Thank you.</div></div><div class="gmail_extra"><br class=""><div class="gmail_quote">2016-05-07 0:30 GMT+08:00 Michael Campbell <span dir="ltr" class=""><<a href="mailto:michael.s.campbell1@gmail.com" target="_blank" class="">michael.s.campbell1@gmail.com</a>></span>:<br class=""><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div style="word-wrap:break-word" class="">So far in the tests that I’ve done I get the same first exon as 5 prime UTR and part of the last exon in 3 prime UTR for that gene.<div class="">Mike<br class=""><div class=""><blockquote type="cite" class=""><div class="">On May 5, 2016, at 10:18 PM, Pei-Ying Huang <<a href="mailto:themis.ray@gmail.com" target="_blank" class="">themis.ray@gmail.com</a>> wrote:</div><br class=""><div class=""><div dir="ltr" class="">Hi Mike,<div class=""><br class=""></div><div class="">I found one five_prime_UTP evidence, but only this one shown in the scaff0001.</div><div class="">Does it mean no more five_prime_UTP on this scaffold or maker doesn't find others?</div><div class="">Thank you.</div><div class=""><br class=""></div><div class=""><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>gene<span style="white-space:pre-wrap" class=""> </span>3190189<span style="white-space:pre-wrap" class=""> </span>3192302<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426;Name=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>mRNA<span style="white-space:pre-wrap" class=""> </span>3190189<span style="white-space:pre-wrap" class=""> </span>3192302<span style="white-space:pre-wrap" class=""> </span>1262<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426;Name=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1;_AED=0.27;_eAED=0.27;_QI=335|0.83|0.71|1|0|0|7|0|308</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>exon<span style="white-space:pre-wrap" class=""> </span>3190189<span style="white-space:pre-wrap" class=""> </span>3190216<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:exon:6;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>exon<span style="white-space:pre-wrap" class=""> </span>3190331<span style="white-space:pre-wrap" class=""> </span>3190656<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:exon:5;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>exon<span style="white-space:pre-wrap" class=""> </span>3190818<span style="white-space:pre-wrap" class=""> </span>3190955<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:exon:4;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>exon<span style="white-space:pre-wrap" class=""> </span>3191233<span style="white-space:pre-wrap" class=""> </span>3191510<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:exon:3;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>exon<span style="white-space:pre-wrap" class=""> </span>3191634<span style="white-space:pre-wrap" class=""> </span>3191666<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:exon:2;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>exon<span style="white-space:pre-wrap" class=""> </span>3191755<span style="white-space:pre-wrap" class=""> </span>3191848<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:exon:1;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>exon<span style="white-space:pre-wrap" class=""> </span>3191938<span style="white-space:pre-wrap" class=""> </span>3192302<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:exon:0;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span><font color="#0000ff" class="">five_prime_UTR</font><span style="white-space:pre-wrap" class=""> </span>3191968<span style="white-space:pre-wrap" class=""> </span>3192302<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:five_prime_utr;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>CDS<span style="white-space:pre-wrap" class=""> </span>3191938<span style="white-space:pre-wrap" class=""> </span>3191967<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>0<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>CDS<span style="white-space:pre-wrap" class=""> </span>3191755<span style="white-space:pre-wrap" class=""> </span>3191848<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>0<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>CDS<span style="white-space:pre-wrap" class=""> </span>3191634<span style="white-space:pre-wrap" class=""> </span>3191666<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>2<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>CDS<span style="white-space:pre-wrap" class=""> </span>3191233<span style="white-space:pre-wrap" class=""> </span>3191510<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>2<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>CDS<span style="white-space:pre-wrap" class=""> </span>3190818<span style="white-space:pre-wrap" class=""> </span>3190955<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>0<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>CDS<span style="white-space:pre-wrap" class=""> </span>3190331<span style="white-space:pre-wrap" class=""> </span>3190656<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>0<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div><div class="">GULI.scaff0001<span style="white-space:pre-wrap" class=""> </span>maker<span style="white-space:pre-wrap" class=""> </span>CDS<span style="white-space:pre-wrap" class=""> </span>3190189<span style="white-space:pre-wrap" class=""> </span>3190216<span style="white-space:pre-wrap" class=""> </span>.<span style="white-space:pre-wrap" class=""> </span>-<span style="white-space:pre-wrap" class=""> </span>1<span style="white-space:pre-wrap" class=""> </span>ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-30.426-mRNA-1</div></div><div class=""><br class=""></div><div class="">Pei-Ying</div></div><div class="gmail_extra"><br class=""><div class="gmail_quote">2016-05-06 8:31 GMT+08:00 Pei-Ying Huang <span dir="ltr" class=""><<a href="mailto:themis.ray@gmail.com" target="_blank" class="">themis.ray@gmail.com</a>></span>:<br class=""><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div dir="ltr" class="">Hi Mike,<div class=""><br class=""></div><div class="">Any clue about the problems?</div><div class="">Or my thought is wrong. I judge the transcript data help or not in maker by checking if est2genome shown in the column 2 in maker output gff file.</div><div class="">Thank you.</div><div class=""><br class=""></div><div class="">Pei-Ying</div><div class=""><br class=""></div></div><div class="gmail_extra"><br class=""><div class="gmail_quote">2016-05-05 1:22 GMT+08:00 Pei-Ying Huang <span dir="ltr" class=""><<a href="mailto:themis.ray@gmail.com" target="_blank" class="">themis.ray@gmail.com</a>></span>:<br class=""><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div dir="ltr" class="">Hi Mike,<div class=""><br class=""></div><div class="">Attached file is the folder I use to run maker. Thank you.</div><div class=""><div class="gmail_chip gmail_drive_chip" style="width:396px;min-height:18px;max-height:18px;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;background-color:rgb(245,245,245)"><a href="https://drive.google.com/file/d/0B1vRN27dO1OBN01reFZLV3JKbGM/view?usp=drive_web" style="display:inline-block;max-width:366px;overflow:hidden;text-overflow:ellipsis;white-space:nowrap;text-decoration:none;padding:1px 0;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="">guliRN_L1_v1_mike.tar.gz</span></a><img style="float:right" class=""></div><br class=""></div><div class="">Pei-Ying</div></div><div class="gmail_extra"><br class=""><div class="gmail_quote">2016-05-04 22:54 GMT+08:00 Michael Campbell <span dir="ltr" class=""><<a href="mailto:michael.s.campbell1@gmail.com" target="_blank" class="">michael.s.campbell1@gmail.com</a>></span>:<br class=""><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"><div style="word-wrap:break-word" class="">Hi Pei-Ying,<div class=""><br class=""></div><div class="">If the sample data didn’t produce est2genome lines when using the sample data then it may be that exonerate is not being called. Could you send me the maker_exe.ctl file.</div><div class=""><br class=""></div><div class="">your maker_opts.ctl file looks fine.</div><div class=""><br class=""></div><div class="">If you have a small test set for your data like a small scaffold that you know has some sringtie hits on it, you could send it to me if you want and I can see if I can figure it out form here if that would be helpful. </div><div class=""><br class=""></div><div class="">Thanks,</div><div class="">Mike<br class=""><div class=""><blockquote type="cite" class=""><div class="">On May 4, 2016, at 12:33 AM, Pei-Ying Huang <<a href="mailto:themis.ray@gmail.com" target="_blank" class="">themis.ray@gmail.com</a>> wrote:</div><br class=""><div class=""><div dir="ltr" class=""><div class="gmail_extra">Hi Mike,</div><div class="gmail_extra"><br class=""></div><div class="gmail_extra"><span style="white-space:nowrap;background-color:rgb(245,245,245)" class="">basic_protocol_1.tar.gz: I run the sample data by </span>Basic protocol 1 in the attached protocol paper uses the drosophila data bundled with MAKER.</div><div class="gmail_extra"><br class=""></div><div class="gmail_extra">I still can't find est2genome in column 2 of gff file and no five_prime_UTR or three_prime_UTR in column 3.</div><div class="gmail_extra"><div class="gmail_extra">I use StringTie to align pair-end reads to genome then use cufflinks2gff to generate the .gff file for maker input.</div><div class="gmail_extra">Since I have three conditions (root, stem, leaf), so I got Root_strtie.gff,Stem_strtie.gff, R_strtie.gff as maker inputs.</div><div class="gmail_extra"><br class=""></div><div class="gmail_extra">Should I merge Root_strtie.gff,Stem_strtie.gff, R_strtie.gff to strtie_merge.gff before input to maker?</div><div class="gmail_extra">When I try to use cufflinks to convert strtie_merge.gtf to strtie_merge.gff, shows the error message below.</div><div class="gmail_extra"><br class=""></div><div class="gmail_extra">/home/pyh/bin/maker/bin/cufflinks2gff3<b class=""> </b>strtie_merge.gtf > strtie_merge.gff</div><div class="gmail_extra"><br class=""></div><div class=""><span style="color:rgb(255,0,0)" class="">Use of uninitialized value $score in join or string at /home/pyh/bin/maker/bin/cufflinks2gff3 line 94, <IN> line 221531.</span></div><div class=""><span style="color:rgb(255,0,0)" class="">Use of uninitialized value $score in join or string at /home/pyh/bin/maker/bin/cufflinks2gff3 line 94, <IN> line 221532.</span></div><div class=""><span style="color:rgb(255,0,0)" class="">Use of uninitialized value $score in join or string at /home/pyh/bin/maker/bin/cufflinks2gff3 line 94, <IN> line 221533.</span></div><div class=""><span style="color:rgb(255,0,0)" class="">Use of uninitialized value $score in join or string at /home/pyh/bin/maker/bin/cufflinks2gff3 line 94, <IN> line 221534.</span></div><div class=""><span style="color:rgb(255,0,0)" class="">Use of uninitialized value $score in join or string at /home/pyh/bin/maker/bin/cufflinks2gff3 line 94, <IN> line 221535.</span></div><div class=""><span style="color:rgb(255,0,0)" class="">Use of uninitialized value $score in join or string at /home/pyh/bin/maker/bin/cufflinks2gff3 line 94, <IN> line 221536.</span></div><div class="gmail_extra"><div class="gmail_chip gmail_drive_chip" style="width:396px;min-height:18px;max-height:18px;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;background-color:rgb(245,245,245)"><a href="https://drive.google.com/file/d/0B1vRN27dO1OBX1djVmpCMHhNT2M/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="">maker1.log</span></a><img style="float:right" class=""></div><div class="gmail_chip gmail_drive_chip" style="width:396px;min-height:18px;max-height:18px;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;background-color:rgb(245,245,245)"><a href="https://drive.google.com/file/d/0B1vRN27dO1OBVWgwcVRmQU1jaW8/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="">maker_opts.log</span></a><img style="float:right" class=""></div><br class=""></div><div class="gmail_extra"><b class="">less A_guli_1.all.gff</b><br class=""></div><div class="gmail_extra">GULI.scaff0001 maker gene 1750118 1755997 . - . ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37;Name=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37</div><div class="gmail_extra">GULI.scaff0001 maker mRNA 1750118 1755997 5292 - . ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37;Name=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1;_AED=0.37;_eAED=0.37;_QI=0|0|0|1|0|0|7|0|1764</div><div class="gmail_extra">GULI.scaff0001 maker exon 1750118 1750214 . - . ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:exon:21;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker exon 1750304 1750815 . - . ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:exon:20;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker exon 1750896 1751717 . - . ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:exon:19;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker exon 1751849 1752373 . - . ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:exon:18;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker exon 1752515 1753488 . - . ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:exon:17;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker exon 1753554 1754406 . - . ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:exon:16;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker exon 1754489 1755997 . - . ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:exon:15;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker CDS 1754489 1755997 . - 0 ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker CDS 1753554 1754406 . - 0 ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker CDS 1752515 1753488 . - 2 ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker CDS 1751849 1752373 . - 0 ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker CDS 1750896 1751717 . - 0 ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker CDS 1750304 1750815 . - 0 ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div><div class="gmail_extra">GULI.scaff0001 maker CDS 1750118 1750214 . - 1 ID=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1:cds;Parent=maker-GULI.scaff0001-exonerate_protein2genome-gene-17.37-mRNA-1</div></div><div class="gmail_extra"><br class=""></div><div class="gmail_extra"><div style="font-size:14px" class="">Thank you.</div><div style="font-size:14px" class="">Pei-Ying</div><div style="font-size:14px" class=""><br class=""></div></div><div class="gmail_extra"><br class=""></div><div class="gmail_extra"><br class=""></div><div class="gmail_extra"><br class=""><div class="gmail_quote">2016-04-14 21:09 GMT+08:00 Michael Campbell <span dir="ltr" class=""><<a href="mailto:michael.s.campbell1@gmail.com" target="_blank" class="">michael.s.campbell1@gmail.com</a>></span>:<br class=""><blockquote class="gmail_quote" style="margin:0px 0px 0px 0.8ex;border-left-width:1px;border-left-color:rgb(204,204,204);border-left-style:solid;padding-left:1ex"><div class="">It is strange for transcripts from the species of interest to not align or help. That FASTA entry looks okay. Did you save the error output from MAKER? if you did could you send it to me along with the MAKER control files? There may be some clues in there. </div><div class=""><br class=""></div><div class="">It would also be good if you could run MAKER on the sample data from drosophila in the /data folder in MAKER. This way we can see if it is your data or your install of MAKER. Basic protocol 1 in the attached protocol paper uses the drosophila data bundled with MAKER.</div><div class=""><br class=""></div><div class="">Aligning with hisat2 and using cufflinks to make transcripts should work. Stringtie seems to have higher specificity than cufflinks and the cufflinks2gff script works on stringtie output as well. You could also do a denovo assembly of the reads yourself using trinity, which has worked well for me in the past. </div><div class=""><br class=""></div><div class="">Protein evidence only will give a reasonable annotation. The transcript data will help in annotating UTRs and species specific genes.</div><div class=""><br class=""></div><div class="">The attached protocol paper also addresses your quality question to an extent.</div></blockquote></div><br class=""><br class=""></div></div>
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