the gene fpkm and isoform fpkm are zero with Cufflink ???

[fulxie]

I used cufflink (v2.0) to analyze my RNA-seq data.
It turned out there was problem with the gene fpkm and isoform fpkm.
The coverage, FPKM, FPKM_conf_lo, and FPKM_conf_hi are zero.
Also, I ran the tophat first and got a BAM file. But the program reported that 'File ./SC/accepted_hits.bam' doesn't appear to be a valid BAM file’.

The reads of my RNA-seq dataset are single-end and are in length of 49nt. My reference genome is consisted of 410,030 contigs

I remember cufflink worked for my zebra fish RNA-seq dataset which is single-end and has reads of 100nt.

Here is the command I used
cufflinks -p 10 -o controlCufflinkOut -g Pvirgatum_202_gene.gff3 ./SC/accepted_hits.bam

I also tried the parameter --library-type fr-firststrand. I got the same result.


Could someone give me some suggestion on this problem?

is there similar tool to replace cufflink?

[sdriscoll]

it depends on whether you need cufflink's transcript assembly abilities or not. i haven't used any other tools for that purpose.

what version of Tophat are you using?

[fulxie]

it depends on whether you need cufflink's transcript assembly abilities or not. i haven't used any other tools for that purpose.

what version of Tophat are you using?

I used the newest tophat v2.0.
I want to assemble it. I thought at least I can get some expression abundances for some annotated genes. I don't know what happened to cufflinks. one of weird is that cufflinks recognized BAM file generated from Tophat as an invalid BAM file

[sdriscoll]

yes, that is weird. that's why i asked what version of Tophat you're using. I'm still on version 1.4 so I'm not sure if your issue could be related to Tophat 2's output or not. the only thing I can suggest is to align the data over again - maybe something went wrong the first time around.

[fulxie]

yes, that is weird. that's why i asked what version of Tophat you're using. I'm still on version 1.4 so I'm not sure if your issue could be related to Tophat 2's output or not. the only thing I can suggest is to align the data over again - maybe something went wrong the first time around.

I turned back to Tophat 1.4 and Cufflinks 1.3 I used on zebrafish before. Unfortunately, it still doesn't work. Do you know there is other tool to do similar analysis as Cufflinks?

[DerSeb]

hmm are the names of your contigs in the annotation gff and the genome the same?

sometimes this happens when your reads map to say chromosome "1" and the gff has only chromosme "chr1"