Brainstorming: Error sources for Cuffquant failure: Map Mass: 0.00

[Thias]

Hello folks,

I have an issue with Cuffquant v2.2.1, which fails to quantify the expression (create .cxb files) of the current RNA-seq I am working on:

 
Inspecting maps and determining fragment length distributions.
Map Properties:
Normalized Map Mass: 0.00
Raw Map Mass: 0.00
Number of Multi-Reads: 0 (with 0 total hits)
Fragment Length Distribution: Truncated Gaussian (default)
Default Mean: 200
Default Std Dev: 80

This is the first paired-end sequenced RNA-seq, which I try to process, however I have a working pipeline for some older single-end Illumina data.

I have doublechecked / considered those error sources and would like to ask you, if you are aware of other possible reasons:

• Reads: Initially quality trimmed, but after I read here in the forum, that this might confuse cufflinks when determining insert sizes, I also aligned the untrimmed reads (adaptors cut) with no luck either.
• Aligner: bbmap 34.41, but I set the options xstag=T and xmtag=T, which is supposed to generate bamfiles suitable for Cufflinks.
• Alignments are indexed. There are clear signals at the positions of the transcripts visible in bedgraph.
• Reference genome and GTF-file: Both have been used for multiple alignments and correctly quantified SE-RNA-seqs. Chromosome names do match. I also unsuccessfully tried another cufflinks-usable GTF-file from a collaborating bioinformatician.
• Although I believe fr-unstranded is the correct library type for the experiment, I tried all three one by one.
• I installed and tried cufflinks v. 2.2.0 instead of v.2.2.1. Both versions successfully were able to requantify a previous RNA-seq and are hence working (same aligner, reference genome and gtf file used)

I would appreciate any suggestions, what I could still try. I am out of ideas at the moment...

Thanks for your help
Matthias