Hello,

I performed 6 different runs of Cufflinks using the same Reference Annotation 
on 6 different samples.  I expected the resulting tabular output to be constant 
given the same Reference Annotation was used each time.  What I found is that 
there was an inconsistency.

Specific example, here is top 5 rows from output for one of the six samples:

tracking_id          class_code          nearest_ref_id gene_id               
gene_short_name          tss_id    locus      length   coverage                
FPKM    FPKM_conf_lo  FPKM_conf_hi  FPKM_status
NM_001005484 -              -              NM_001005484 -              -        
      chr1:69090-70008             -              -              0              
0              0                OK
NR_026820         -              -              NR_026820         -             
 -              chr1:34610-36081             -              -              0    
          0              0                OK
NM_001005221 -              -              NM_001005221 -              -        
      chr1:367658-368597        -              -              0              0  
            0                OK
NR_046018         -              -              NR_046018         -             
 -              chr1:11873-14408             -              -              0    
          0              0                OK

Here are top 5 rows from output for another of the six samples:

tracking_id          class_code          nearest_ref_id gene_id               
gene_short_name          tss_id    locus      length   coverage                
FPKM    FPKM_conf_lo  FPKM_conf_hi  FPKM_status
NM_001005484 -              -              NM_001005484 -              -        
      chr1:69090-70008             -              -              0              
0              0                OK
NR_026820         -              -              NR_026820         -             
 -              chr1:34610-36081             -              -              0    
          0              0                OK
NM_001005221 -              -              NM_001005221 -              -        
      chr1:367658-368597        -              -              0              0  
            0                OK
NR_046018         -              -              NR_046018         -             
 -              chr1:11873-14408             -              -              0    
          0              0                OK
NR_024540         -              -              NR_024540         -             
 -              chr1:14361-29370             -              -              
15114.7 9501.21                20728.1 OK

Here are top 5 rows from output for yet another of the six samples:

tracking_id          class_code          nearest_ref_id gene_id               
gene_short_name          tss_id    locus      length   coverage                
FPKM    FPKM_conf_lo  FPKM_conf_hi  FPKM_status
NR_026820         -              -              NR_026820         -             
 -              chr1:34610-36081             -              -              0    
          0              0                OK
NM_001005484 -              -              NM_001005484 -              -        
      chr1:69090-70008             -              -              0              
0              0                OK
NM_001005221 -              -              NM_001005221 -              -        
      chr1:367658-368597        -              -              0              0  
            0                OK
NR_046018         -              -              NR_046018         -             
 -              chr1:11873-14408             -              -              0    
          0              0                OK
NR_024540         -              -              NR_024540         -             
 -              chr1:14361-29370             -              -              
28376.8 20008.9                36744.6 OK

As a user, when selecting the same Reference Annotation to be used.  I would 
expect the index of results by tracking id to be the same.

Why is this happening?  Why are they not consistent?

If  were to concat the above results across samples without correcting the 
indices, my data would be all mixed up.

I bring this to your attention as someone may not correct the indices before 
concat and therefore would be doing downstream analysis incorrect concatted 
data.

In turn, I would recommend that the index of results be corrected to be 
constant for a Reference Annotation when selected.

Thank you in advance for this consideration.

Best,

Kory Johnson

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

Kory R. Johnson, MS, PhD
Sr. Bioinformatics Scientist

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