Question: Cufflinks produces empty transcript.gtf files
gravatar for william.mcentegart
2.9 years ago by
william.mcentegart0 wrote:

Hi there. I have cufflinks running without errors except it produces empty transcript.gtf files. I am aliging reads with Bowtie2 and converting sam into bam and indexing with samtools. I then take the indexed file (.sorted) into cufflinks with my fasta file and gff file. The GFF and the fasta have the correct headings (there is no separate chromosomes).

Using this command

/opt/cufflinks/cufflinks -o cufflinks/sample.gff -g genome.gff -p8 -b genome.fasta -u --library-type fr-unstranded sample.sorted --verbose

I get a bunch of warnings like this

Warning: CDS ORF_02239 found without exon segments (adding default exon).
Warning: CDS ORF_02240 found without exon segments (adding default exon).
Warning: CDS ORF_02241 found without exon segments (adding default exon).
Warning: CDS ORF_02242 found without exon segments (adding default exon).

Followed by

Kept 2279 transfrags out of 2279
1: null
[14:54:58] Inspecting reads and determining fragment length distribution.
Inspecting bundle null:0-2253459 with 9257857 reads
Processed 1 loci.
Warning: Using default Gaussian distribution due to insufficient paired-end reads in open ranges.  It is recommended that correct parameters (--frag-len-mean and --frag-len-std-dev) be provided.
> Map Properties:
>       Normalized Map Mass: 9296492.50
>       Raw Map Mass: 9296492.50
>       Number of Multi-Reads: 0 (with 0 total hits)
>       Fragment Length Distribution: Truncated Gaussian (default)
>                     Default Mean: 200
>                  Default Std Dev: 80
0 ReadHits still live
Found 2 reference contigs
        Total map density: 9296492.500000
[14:58:45] Assembling transcripts and initializing abundances for multi-read correction.
null:0-2253459  Processing new bundle with 9257857 alignments
Processed 1 loci.
[15:02:34] Loading reference annotation and sequence.
 Kept 0 transfrags out of 0
[15:02:34] Learning bias parameters.
Processed 0 loci.
[15:03:05] Re-estimating abundances with bias and multi-read correction.
Processed 0 loci.

Please can i have some help im at a brick wall.


bam rna-seq cufflinks bowtie2 fasta • 1.1k views
ADD COMMENTlink written 2.9 years ago by william.mcentegart0

Hopefully the bams also have the correct chromosome names. gffread command (from cufflinks package) on the gff could be tried to check the gff. A flagstat on the bams might also be useful to diagnose.

ADD REPLYlink written 2.9 years ago by microfuge1.5k
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