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Old 01-26-2016, 09:03 AM   #1
ivygreen
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Default Reads Mapped but 0 FPKM for Every Gene

Hi all,

I'm using Tophat 2.1.0 and Cufflinks 2.2.1 to analyze mouse RNA sequencing. I used Tophat to align the reads, and this step appeared to be successful. A sample alignment summary for one of my Tophat runs is below:

--------------------------------------

>008_F0/align_summary.txt
Left reads:
Input : 16203854
Mapped : 12585562 (77.7% of input)
of these: 7295254 (58.0%) have multiple alignments (38724 have >20)
Right reads:
Input : 16203854
Mapped : 11407353 (70.4% of input)
of these: 6601221 (57.9%) have multiple alignments (38723 have >20)
74.0% overall read mapping rate.

Aligned pairs: 10988017
of these: 6368400 (58.0%) have multiple alignments
1480 ( 0.0%) are discordant alignments
67.8% concordant pair alignment rate.

--------------------------------------

However, when I use cuffdiff to get abundance and differential expression estimates, I get 0 FPKM for everything (all the values for the files created by this step have 0 values!).

I suspect that the error is in the original cuffdiff step, but I'm not sure what is wrong. My reference files are from http://ftp.ensembl.org/pub/current_fasta/. This was the output for my cuffdiff run:

--------------------------------------

>cuffdiff -o all-diffs -b download/Mus_musculus.GRCm38.dna.toplevel.fa -p 4 -L 008_F0,008_F4,018_F0,018_F4,019_F0,019_F4 -u download/Mus_musculus.GRCm38.83.gtf 008_F0/accepted_hits.bam 008_F4/accepted_hits.bam 018_F0/accepted_hits.bam 018_F4/accepted_hits.bam 019_F0/accepted_hits.bam 019_F4/accepted_hits.bam

You are using Cufflinks v2.2.1, which is the most recent release.
[16:08:46] Loading reference annotation and sequence.
Warning: No conditions are replicated, switching to 'blind' dispersion method
[16:09:41] Inspecting maps and determining fragment length distributions.
[16:19:10] Modeling fragment count overdispersion.
> 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
> 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
> 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
> 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
> 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
> 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
[16:25:17] Calculating preliminary abundance estimates
> Processed 33596 loci. [*************************] 100%
[16:51:51] Learning bias parameters.
[17:00:27] Testing for differential expression and regulation in locus.
> Processed 33596 loci. [*************************] 100%
Performed 0 isoform-level transcription difference tests
Performed 0 tss-level transcription difference tests
Performed 0 gene-level transcription difference tests
Performed 0 CDS-level transcription difference tests
Performed 0 splicing tests
Performed 0 promoter preference tests
Performing 0 relative CDS output tests
Writing isoform-level FPKM tracking
Writing TSS group-level FPKM tracking
Writing gene-level FPKM tracking
Writing CDS-level FPKM tracking
Writing isoform-level count tracking
Writing TSS group-level count tracking
Writing gene-level count tracking
Writing CDS-level count tracking
Writing isoform-level read group tracking
Writing TSS group-level read group tracking
Writing gene-level read group tracking
Writing CDS-level read group tracking
Writing read group info
Writing run info

--------------------------------------

As you can see, all the map properties were 0, which I was told might not be an incorrect result, although it seemed unexpected to me. Does anyone know of anything else that I can try? Any suggestions would be greatly appreciated.

Thanks in advance!
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Old 01-26-2016, 11:35 AM   #2
westerman
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Looks like you are doing things ok. But try without the '-u' option.
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Old 01-26-2016, 06:24 PM   #3
ivygreen
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I tried re-running without the -u option, but all FPKM are still 0.
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Old 01-27-2016, 03:37 AM   #4
Michael.Ante
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Hi Ivygreen,
I'd run cufflinks on one/two sample first, there you'll maybe get a more detailed report. Check also if the bam-files are indexed (e.g. samtools index 008_F0/accepted_hits.bam) and if the chromosome names are the same in the alignment and the GTF file.
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Old 05-24-2016, 11:24 AM   #5
ivygreen
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Hi Michael Ante,
I'm pretty new to these programs. I ran cufflinks on a few samples - how do I interpret the resulting reports? Also, if the files are indexed with different alignments (which I now suspect they are), is there an easy way to re-align them properly?
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Old 05-24-2016, 11:58 AM   #6
cmbetts
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This wouldn't explain why your FPKMs are zero for every gene, but your alignment statistics are a little alarming.
Aligned pairs: 10988017
of these: 6368400 (58.0%) have multiple alignments
That's a really high multiple alignment rate for mouse RNA-Seq. I've only ever seen it that high when the rRNA depletion/ polyA selection failed. Even so, with 10M reads, you should be picking up >10k genes. How were these samples prepared?
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Old 05-24-2016, 10:47 PM   #7
Michael.Ante
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Hi Ivygreen,

did you receive the same map mass as with cuffdiff? If not, you can use e.g. R to analyse the genes.fpkm_tracking and isoforms.fpkm_tracking files. For instance, you can make a boxplot of the FPKM values.

Could you please post the tophat command you were using for the alignment?

Cheers,

Michael
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