<div dir="ltr">Dear Developer,<div><br></div><div>I was trying to use a mixed GFF3 file to do some analysis, which includes genome-level gene predictions and manually curated annotations. I replaced the gene prediction by Maker annotations where appropriate. And a sample of the blended GFF3 file is as follows:</div><div><br></div><div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">  </span>GLEAN<span class="Apple-tab-span" style="white-space:pre">       </span>mRNA<span class="Apple-tab-span" style="white-space:pre">        </span>3918<span class="Apple-tab-span" style="white-space:pre">        </span>18376<span class="Apple-tab-span" style="white-space:pre">       </span>0.920154<span class="Apple-tab-span" style="white-space:pre">    </span>+<span class="Apple-tab-span" style="white-space:pre">   </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>ID=CCG000529.1;source_id=Aalb_GLEAN_10002648;</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">   </span>GLEAN<span class="Apple-tab-span" style="white-space:pre">       </span>CDS<span class="Apple-tab-span" style="white-space:pre"> </span>3918<span class="Apple-tab-span" style="white-space:pre">        </span>3983<span class="Apple-tab-span" style="white-space:pre">        </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>+<span class="Apple-tab-span" style="white-space:pre">   </span>0<span class="Apple-tab-span" style="white-space:pre">   </span>Parent=CCG000529.1;</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">     </span>GLEAN<span class="Apple-tab-span" style="white-space:pre">       </span>CDS<span class="Apple-tab-span" style="white-space:pre"> </span>4521<span class="Apple-tab-span" style="white-space:pre">        </span>4598<span class="Apple-tab-span" style="white-space:pre">        </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>+<span class="Apple-tab-span" style="white-space:pre">   </span>0<span class="Apple-tab-span" style="white-space:pre">   </span>Parent=CCG000529.1;</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">     </span>GLEAN<span class="Apple-tab-span" style="white-space:pre">       </span>CDS<span class="Apple-tab-span" style="white-space:pre"> </span>17407<span class="Apple-tab-span" style="white-space:pre">       </span>17516<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>+<span class="Apple-tab-span" style="white-space:pre">   </span>0<span class="Apple-tab-span" style="white-space:pre">   </span>Parent=CCG000529.1;</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">     </span>GLEAN<span class="Apple-tab-span" style="white-space:pre">       </span>CDS<span class="Apple-tab-span" style="white-space:pre"> </span>17588<span class="Apple-tab-span" style="white-space:pre">       </span>17767<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>+<span class="Apple-tab-span" style="white-space:pre">   </span>1<span class="Apple-tab-span" style="white-space:pre">   </span>Parent=CCG000529.1;</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">     </span>GLEAN<span class="Apple-tab-span" style="white-space:pre">       </span>CDS<span class="Apple-tab-span" style="white-space:pre"> </span>18238<span class="Apple-tab-span" style="white-space:pre">       </span>18376<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>+<span class="Apple-tab-span" style="white-space:pre">   </span>1<span class="Apple-tab-span" style="white-space:pre">   </span>Parent=CCG000529.1;</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">     </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>gene<span class="Apple-tab-span" style="white-space:pre">        </span>32452<span class="Apple-tab-span" style="white-space:pre">       </span>54508<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0;Name=AAEL004146</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">    </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>mRNA<span class="Apple-tab-span" style="white-space:pre">        </span>32452<span class="Apple-tab-span" style="white-space:pre">       </span>54508<span class="Apple-tab-span" style="white-space:pre">       </span>44204<span class="Apple-tab-span" style="white-space:pre">       </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0;Name=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2;aED=0.00;eAED=0.00;qI=376|1|1|1|0|0|5|316|548</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">        </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>exon<span class="Apple-tab-span" style="white-space:pre">        </span>32452<span class="Apple-tab-span" style="white-space:pre">       </span>33011<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2:exon:15;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">     </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>exon<span class="Apple-tab-span" style="white-space:pre">        </span>35140<span class="Apple-tab-span" style="white-space:pre">       </span>35388<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1:exon:12;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1,maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">    </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>exon<span class="Apple-tab-span" style="white-space:pre">        </span>35455<span class="Apple-tab-span" style="white-space:pre">       </span>36119<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1:exon:11;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1,maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">    </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>exon<span class="Apple-tab-span" style="white-space:pre">        </span>36443<span class="Apple-tab-span" style="white-space:pre">       </span>36777<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1:exon:10;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1,maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">    </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>exon<span class="Apple-tab-span" style="white-space:pre">        </span>53979<span class="Apple-tab-span" style="white-space:pre">       </span>54508<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1:exon:9;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1,maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">     </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>five_prime_UTR<span class="Apple-tab-span" style="white-space:pre">      </span>54133<span class="Apple-tab-span" style="white-space:pre">       </span>54508<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2:five_prime_utr;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre">      </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>CDS<span class="Apple-tab-span" style="white-space:pre"> </span>53979<span class="Apple-tab-span" style="white-space:pre">       </span>54132<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>0<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2:cds;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre"> </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>CDS<span class="Apple-tab-span" style="white-space:pre"> </span>36443<span class="Apple-tab-span" style="white-space:pre">       </span>36777<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>2<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2:cds;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre"> </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>CDS<span class="Apple-tab-span" style="white-space:pre"> </span>35455<span class="Apple-tab-span" style="white-space:pre">       </span>36119<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>0<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2:cds;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre"> </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>CDS<span class="Apple-tab-span" style="white-space:pre"> </span>35140<span class="Apple-tab-span" style="white-space:pre">       </span>35388<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>1<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2:cds;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre"> </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>CDS<span class="Apple-tab-span" style="white-space:pre"> </span>32768<span class="Apple-tab-span" style="white-space:pre">       </span>33011<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>1<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2:cds;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div><div>scaffold10370<span class="Apple-tab-span" style="white-space:pre"> </span>maker<span class="Apple-tab-span" style="white-space:pre">       </span>three_prime_UTR<span class="Apple-tab-span" style="white-space:pre">     </span>32452<span class="Apple-tab-span" style="white-space:pre">       </span>32767<span class="Apple-tab-span" style="white-space:pre">       </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>-<span class="Apple-tab-span" style="white-space:pre">   </span>.<span class="Apple-tab-span" style="white-space:pre">   </span>ID=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2:three_prime_utr;Parent=maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-2</div></div><div><br></div>This file does not pass the GFF3 online validator from GenomeTools with the following error message:<div><br>GenomeTools error: Parent "maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1" on line 1456 in file "/var/www/servers/<a href="http://genometools.org/htdocs/cgi-bin/gff3/Aalbo.gff">genometools.org/htdocs/cgi-bin/gff3/Aalbo.gff</a>" was not defined (via "ID=")</div><div><br></div><div>And when I tried to use gff3toGenePred to convert GFF3 to the refflat format, I got the following parsing errors (samples):</div><div><br></div>Can't find annotation record "maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1" referenced by "maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1:exon:12" Parent attribute<br><br>Can't find annotation record "maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1" referenced by "maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1:exon:11" Parent attribute<br><br>Can't find annotation record "maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1" referenced by "maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1:exon:10" Parent attribute<br><br>Can't find annotation record "maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1" referenced by "maker-scaffold10370-exonerate_est2genome-gene-0.0-mRNA-1:exon:9" Parent attribute<br><br>Can't find annotation record "maker-scaffold1193-exonerate_est2genome-gene-2.0-mRNA-2" referenced by "maker-scaffold1193-exonerate_est2genome-gene-2.0-mRNA-1:exon:114" Parent attribute<br><br>Can't find annotation record "maker-scaffold16459-exonerate_est2genome-gene-0.0-mRNA-1" referenced by "maker-scaffold16459-exonerate_est2genome-gene-0.0-mRNA-1:exon:118" Parent attribute<br><br>Can't find annotation record "maker-scaffold16459-exonerate_est2genome-gene-0.0-mRNA-1" referenced by "maker-scaffold16459-exonerate_est2genome-gene-0.0-mRNA-1:exon:119" Parent attribute<br><br>Can't find annotation record "maker-scaffold16459-exonerate_est2genome-gene-0.0-mRNA-1" referenced by "maker-scaffold16459-exonerate_est2genome-gene-0.0-mRNA-1:exon:120" Parent attribute<br><br>Can't find annotation record "maker-scaffold16459-exonerate_est2genome-gene-0.0-mRNA-1" referenced by "maker-scaffold16459-exonerate_est2genome-gene-0.0-mRNA-1:exon:121" Parent attribute<div><br></div><div>What could have been the issue in the annotation file or the way I used the file? Any feedback will be highly appreciated!</div><div><br></div><div>Sincerely,</div><div><br></div><div>Xin Huang</div></div>