<html><head><style>body{font-family:Helvetica,Arial;font-size:13px}</style></head><body style="word-wrap:break-word"><div id="bloop_customfont" style="font-family:Helvetica,Arial;font-size:13px;color:rgba(0,0,0,1.0);margin:0px;line-height:auto"><p style="margin:15px 0px;font-family:'helvetica Neue',helvetica;font-size:14px">Hi dev@ensembl members,</p><p style="margin:15px 0px;font-family:'helvetica Neue',helvetica;font-size:14px">I investigate a problem regarding cuffmerge in combination with ensembl GFF3 files and seek help in understanding the difference between the GFF3 and GTF files in ensembl.</p><p style="margin:15px 0px;font-family:'helvetica Neue',helvetica;font-size:14px">I align RNA-Seq reads with HISAT2 to the reference genome and then derive the transcriptome by running cufflinks with the Mus.Musculus e.86 release <em>GFF3</em> file. When I run cuffmerge on these files it fails with an error <code style="font-family:Menlo,Consolas,'Liberation Mono',Courier,monospace;font-size:10pt;border-top-left-radius:3px;border-top-right-radius:3px;border-bottom-right-radius:3px;border-bottom-left-radius:3px;background-color:rgb(248,248,248);color:inherit;border:1px solid rgb(234,234,234);margin:0px 2px;padding:0px 5px;word-break:normal;word-wrap:normal">GFF Error: duplicate/invalid 'transcript' feature ID=transcript:ENSMUST00000045689</code>.</p><p style="margin:15px 0px;font-family:'helvetica Neue',helvetica;font-size:14px">When I do the very same analysis with the Mus.Musculus e.86 release <em>GTF</em> file, everything runs fine. I investigated the<code style="font-family:Menlo,Consolas,'Liberation Mono',Courier,monospace;font-size:10pt;border-top-left-radius:3px;border-top-right-radius:3px;border-bottom-right-radius:3px;border-bottom-left-radius:3px;background-color:rgb(248,248,248);color:inherit;border:1px solid rgb(234,234,234);margin:0px 2px;padding:0px 5px;word-break:normal;word-wrap:normal">ENSMUST00000045689</code> transcript and indeed found differences between the GTF and GFF3 file! This is potentially causing the problem in cufflinks.</p><p style="margin:15px 0px;font-family:'helvetica Neue',helvetica;font-size:14px">The description of the difference and a fully functional minimal example can be found in this repository: <a href="https://github.com/paulklemm/cuffmerge_bug" style="color:rgb(65,131,196);background-color:inherit;text-decoration:none">https://github.com/paulklemm/cuffmerge_bug</a>.</p><p style="margin:15px 0px;font-family:'helvetica Neue',helvetica;font-size:14px">My question is: Why is there a difference in the annotation between the GFF3 and GTF files? I thought that it is the same information just stored in different formats. That seems not to be the case.</p><p style="margin:15px 0px;font-family:'helvetica Neue',helvetica;font-size:14px">I contributed the code to a recent bug report in the cufflinks repository, which describes this problem: <a href="https://github.com/cole-trapnell-lab/cufflinks/issues/77" style="color:rgb(65,131,196);background-color:inherit;text-decoration:none">https://github.com/cole-trapnell-lab/cufflinks/issues/77</a>.</p><p style="margin:15px 0px;font-family:'helvetica Neue',helvetica;font-size:14px">Thanks for the help.</p><p style="margin:15px 0px;font-family:'helvetica Neue',helvetica;font-size:14px">Paul</p></div><br><div id="bloop_sign_1477488549583836160" class="bloop_sign"></div></body></html>