<p>I am trying to get tophat / cufflinks to work with Ensembl annotations. </p>

<p>Although this looks like a long post, my problem is quite simple: I keep getting <strong>CUFF.1</strong>, <strong>CUFF.2</strong>, <strong>CUFF.3</strong>, etc, as my gene<em>id, in the genes.fpkm</em>tracking file:</p>

<pre><code>$ head genes.fpkm_tracking 
tracking_id     class_code      nearest_ref_id  gene_id gene_short_name tss_id  locus   length  coverage        FPKM    FPKM_conf_lo    FPKM_conf_hi    FPKM_status
CUFF.1  -       -       CUFF.1  -       -       chr1:568966-570302      -       -       1080.24 1019.13 1141.36 OK
CUFF.4  -       -       CUFF.4  -       -       chr1:979748-982084      -       -       19.74   10.1647 29.3153 OK
CUFF.2  -       -       CUFF.2  -       -       chr1:982297-984413      -       -       11.6648 5.19434 18.1353 OK
CUFF.3  -       -       CUFF.3  -       -       chr1:881909-892399      -       -       24.6448 15.3299 33.9596 OK
</code></pre>

<p>What I want is to have the Ensembl ENSG annotations, and possibly gene_name as well.</p>

<p>Yes, I realize the chromosome annotation (1st column) in the GTF/GFF file must match the FASTA header exactly.  </p>

<p>I have tried the "native" Ensembl GTF file (chromosomes numbered 1, 2, 3..., X, Y, MT) and a bowtie2 genome index built from Ensembl's GRCh37.69 FASTA:</p>

<pre><code>1       processed_transcript    exon    11869   12227   .       +       .        gene_id "ENSG00000223972"; transcript_id "ENST00000456328"; exon_number "1"; gene_name "DDX11L1"; gene_biotype "pseudogene"; transcript_name "DDX11L1-002"; exon_id "ENSE00002234944";
1       processed_transcript    exon    12613   12721   .       +       .        gene_id "ENSG00000223972"; transcript_id "ENST00000456328"; exon_number "2"; gene_name "DDX11L1"; gene_biotype "pseudogene"; transcript_name "DDX11L1-002"; exon_id "ENSE00002867822";
1       processed_transcript    exon    13221   14409   .       +       .        gene_id "ENSG00000223972"; transcript_id "ENST00000456328"; exon_number "3"; gene_name "DDX11L1"; gene_biotype "pseudogene"; transcript_name "DDX11L1-002"; exon_id "ENSE00002312635";
</code></pre>

<p>I have tried the native GTF with "chr" added in, and with the FASTA file modified in the same way (and of course bt2 index rebuilt):</p>

<pre><code>chr1    processed_transcript    exon    11869   12227   .       +       .        gene_id "ENSG00000223972"; transcript_id "ENST00000456328"; exon_number "1"; gene_name "DDX11L1"; gene_biotype "pseudogene"; transcript_name "DDX11L1-002"; exon_id "ENSE00002234944";
chr1    processed_transcript    exon    12613   12721   .       +       .        gene_id "ENSG00000223972"; transcript_id "ENST00000456328"; exon_number "2"; gene_name "DDX11L1"; gene_biotype "pseudogene"; transcript_name "DDX11L1-002"; exon_id "ENSE00002867822";
chr1    processed_transcript    exon    13221   14409   .       +       .        gene_id "ENSG00000223972"; transcript_id "ENST00000456328"; exon_number "3"; gene_name "DDX11L1"; gene_biotype "pseudogene"; transcript_name "DDX11L1-002"; exon_id "ENSE00002312635";
</code></pre>

<p>I have also tried using cufflinks gffread utility, to make a GFF file from the above GTF file:</p>

<pre><code>chr1    processed_transcript    gene    11869   14412   .       +       .       ID=ENSG00000223972;Name=DDX11L1;transcripts=ENST00000450305,ENST00000456328,ENST00000515242,ENST00000518655
chr1    processed_transcript    misc_RNA        11869   14409   .       +       .       ID=ENST00000456328;Parent=ENSG00000223972;geneID=ENSG00000223972;gene_name=DDX11L1;Name=DDX11L1-002;type=pseudogene
chr1    processed_transcript    exon    11869   12227   .       +       .       Parent=ENST00000456328
chr1    processed_transcript    exon    12613   12721   .       +       .       Parent=ENST00000456328
chr1    processed_transcript    exon    13221   14409   .       +       .       Parent=ENST00000456328
</code></pre>

<p>For which the corresponding bowtie2 index has these entries:</p>

<pre><code>$ bowtie2-inspect -n /hulk/genomes/Ensembl/Homo_sapiens.GRCh37.69.dna.chromosome 
chr10
chr11
chr12
chr13
...
</code></pre>

<p>I have even tried using the iGenomes files, with the same results!</p>

<p>The way I am running tophat and cufflinks is like this:</p>

<pre><code>tophat -G Homo_sapiens.GRCh37.69.gtf --transcriptome-index=Homo_sapiens.GRCh37.69.genes -p 24 -o output Homo_sapiens.GRCh37.69.dna Sample1_1.fq.gz Sample1_2.fq.gz
cufflinks -p 6 -o output output/accepted_hits.bam
</code></pre>

<p>It is almost like I am running it without annotations, but the tophat / cufflinks programs never complain about being unable to match annotations with the FASTA file or anything:</p>

<pre><code>[2012-11-09 17:11:40] Beginning TopHat run (v2.0.6)
-----------------------------------------------
[2012-11-09 17:11:40] Checking for Bowtie
                  Bowtie version:        2.0.2.0
[2012-11-09 17:11:40] Checking for Samtools
                Samtools version:        0.1.18.0
[2012-11-09 17:11:41] Checking for Bowtie index files
[2012-11-09 17:11:41] Checking for reference FASTA file
[2012-11-09 17:11:41] Generating SAM header for /hulk/genomes/Ensembl/Homo_sapiens.GRCh37.69.dna.chromosome
        format:          fastq
        quality scale:   phred33 (default)
[2012-11-09 17:11:44] Reading known junctions from GTF file
[2012-11-09 17:12:02] Preparing reads
         left reads: min. length=30, max. length=101, 999911 kept reads (89 discarded)
        right reads: min. length=30, max. length=101, 999341 kept reads (659 discarded)
[2012-11-09 17:12:43] Creating transcriptome data files..
...snip...
[2012-11-09 18:10:58] Reporting output tracks
-----------------------------------------------
[2012-11-09 18:16:25] Run complete: 01:04:44 elapsed

You are using Cufflinks v2.0.2, which is the most recent release.
[18:16:26] Inspecting reads and determining fragment length distribution.
&gt; Processed 120637 loci.                       [*************************] 100%
&gt; Map Properties:
&gt;       Normalized Map Mass: 985482.42
&gt;       Raw Map Mass: 985482.42
&gt;       Fragment Length Distribution: Empirical (learned)
&gt;                     Estimated Mean: 163.12
&gt;                  Estimated Std Dev: 50.10
[18:17:15] Assembling transcripts and estimating abundances.
&gt; Processed 120951 loci.                       [*************************] 100%
</code></pre>

<p>Yes, this topic has been discussed online ad nauseum.  <a href="http://seqanswers.com/forums/archive/index.php/t-12694.html">Here</a>, and <a href="http://cufflinks.cbcb.umd.edu/gff.html">here</a>, and <a href="http://www.biostars.org/p/52016/">here</a>, and many other places.  I feel like I have read every possible internet page about this topic, but still the problem vexes me.  In the past I once had this working just fine.  In fact, I found that tophat / cufflinks worked very nicely with Ensembl annotations.  However, it seems like something has changed -- either the software or the annotations?  Am I the only one experiencing this?  Can someone help me see what I am overlooking?  Any help would be much appreciated.</p>

<p>Here are the software versions I am using:</p>

<pre><code>$ bowtie2 --version
/usr/local/bin/bowtie2-2.0.2/bowtie2-align version 2.0.2
64-bit
Built on igm1
Wed Oct 31 23:16:47 EDT 2012
$ tophat --version
TopHat v2.0.6
$ cufflinks 
cufflinks v2.0.2
linked against Boost version 104900
</code></pre>

<p>It doesn't appear that you're setting either the -G/--GTF or the -g/--GTF-guide flags when you run cufflinks. See comments above as well.</p>
