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Old 10-11-2011, 07:54 AM   #1
Location: Italy

Join Date: Jul 2010
Posts: 27
Default Cuffcompare/cuffdiff changes FPKM values of same BAM in sequential runs

Dear all,

Question 1.

For an RNA Seq experiment I compare between 3 bam files (non paired-end). (Tophat- Cufflinks)

B vs A , C vs A (no replicate arrays)

In the first step : sequentially run Tophat-Cufflinks accross each of my files to create all the bam files.

In the second step: sequentially run comparisons with cuffcompare-cuffdiff.

For most XLOCs the FPKM of "setA" differs slightly from one comparison to the next as in the example below.
Why might that be? it is the same bam file in both cases. In certain rare cases the FPKM of "setA" differs considerably accross the comparisons.

Comparison B vs A

XLOC_002887 setB setA NOTEST 200,206 186,633
XLOC_003705 setB setA LOWDATA 152,669 223,705

Comparison C vs A

XLOC_002887 setC setA LOWDATA 201,762 185,595
XLOC_003705 setC setA LOWDATA 253,098 222,461

The basic workflow with options where applicable follows

Tophat -i 30 -I 20000 --segment-length 16 --segment-mismatches 1 (since my reads are 32bp long I have used half of the read length)
Cufflinks -N
Cuffdiff -N -L B,A --FDR 0.1

Question 2.

In a replay of this experiment as an exercise I used instead of "--segment-length 16" , "--segment-length 20" which is more than half the length of my reads. In this case , ALL FPMKs were similar to the 16 segment case but multiplied by 100

How can this happen?

Thanks for your input.
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