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

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Kory R. Johnson, MS, PhD

Sr. Bioinformatics Scientist

www.kellygovernmentsolutions.com

Providing Contract Services For:

Bioinformatics Section,

Information Technology & Bioinformatics Program,

Division of Intramural Research (DIR),

National Institute of Neurological Disorders & Stroke (NINDS),

National Institutes of Health (NIH),

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