Warning: Using default Gaussian distribution due to insufficient paired-end reads in open ranges

Question

[mike]

Hi,

I am doing cuffdiff analysis for case and control study with 3 biological replicates each. I have paired end 100bp reads from Illumina and I got the following warning message.

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.

The gene_exp.diff looks like following. There are no significant genes at all!

test_id gene_id gene    locus   sample_1        sample_2        status  value_1 value_2 log2(fold_change)       test_stat       p_value q_value significant
CUFF.1  CUFF.1  -       1:141473-149707 CASE    CONTROL NOTEST  0       0       0       0       1       1       no
CUFF.10 CUFF.10 -       1:803450-812283 CASE    CONTROL NOTEST  0       0       0       0       1       1       no

Here are the parameters which I have used for assembling, merging transcripts and differential expression analysis.

cufflinks -p 4 -g genes.gtf -o output_cl input.bam
cuffmerge -p 4 -o merged -g genes.gtf -s genome.fa assemblies.txt
cuffdiff -p 4 -o cuffdiff_out -L CASE,CONTROL -u merged/merged.gtf \
rep1.bam,rep2.bam,rep3.bam \
rep1_1.bam,rep2_2.bam,rep3_3.bam

Thanks

Answer 1

[Devon Ryan]

Do you have paired-end reads? If not, you'll always get that message, which you can ignore.

The other issue is that I don't think cuff diff supports case control designs. You're actually comparing two groups, so if you truly have a paired design then you're over-estimating the variance.