Control-FREEC: mouse WES data, with control

[morrowliu]

Hi,

I am using control-FREEC to analyze a few mouse WES samples to get the CNVs.
I went through the manual and created config_ctrl file as suggested in the manual and the previous posts here, but I still encounter one error message and I couldn't figure out why.
Greatly appreciate if anyone can help me with it. Thanks a lot in advance!

Control-FREEC v7.2 : calling copy number alterations and LOH regions using deep-sequencing data
MT-mode using 6 threads
..Minimal CNA length (in windows) is 4
..Breakpoint threshold for segmentation of copy number profiles is 0.8
..Polynomial degree for "ReadCount ~ GC-content" or "Sample ReadCount ~ Control ReadCount" is 3
..telocenromeric set to 50000
..FREEC is not going to output normalized copy number profiles into a BedGraph file (for example, for visualization in the UCSC GB). Use "[general] BedGraphOutput=TRUE" if you want a BedGraph file
..FREEC is going to adjust profiles for a possible contamination by normal cells
..set contaminationAdjustment=FALSE if you don't want to use this option because you think that there is no contamiantion of your tumor sample by normal cells (e.g., it is a cell line, or it non-cancer DNA used without a control sample)
..FREEC is going to evaluate contamination by normal cells
..Note, the Coefficient Of Variation won't be used since "window" = 8000 was set
..Step:	2500
..Output directory:	/rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/FreeC/pilot/working/results/
..Sample file:	/rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/sequenza/pilot/0001-samtools-prep/output/S08.pileup.gz
..Sample input format:	pileup
..Control file:	/rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/sequenza/pilot/0001-samtools-prep/output/S01.pileup.gz
..Input format for the control file:	pileup
..File with chromosome lengths:	/rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/FreeC/pilot/control/mouse_chr_y.len
..uniqueMatch = FALSE
..average ploidy set to 2
..break-point type set to 4
Warning: Parameter '[general] noisyData=true' will not have effect since FREEC won't use BAF information to correct predicted copy numbers
..minimal number of reads per window in the control sample is set to 50
..File /rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/FreeC/pilot/control/mouse_chr_y.len was read
..Starting reading /rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/sequenza/pilot/0001-samtools-prep/output/S08.pileup.gz
PROFILING [tid=140174374557472]: /rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/sequenza/pilot/0001-samtools-prep/output/S08.pileup.gz read in 155 seconds [fillMyHash]
260591386 lines read..
47399307 reads used to compute copy number profile
printing counts into /rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/FreeC/pilot/working/results/S08.pileup.gz_sample.cpn
..Window size:	8000
..File /rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/FreeC/pilot/control/mouse_chr_y.len was read
..Starting reading /rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/sequenza/pilot/0001-samtools-prep/output/S01.pileup.gz
PROFILING [tid=140174374557472]: /rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/sequenza/pilot/0001-samtools-prep/output/S01.pileup.gz read in 202 seconds [fillMyHash]
278748064 lines read..
68986773 reads used to compute copy number profile
printing counts into /rsrch1/epi/scheet/PROJECTS/Kadara_Mouse/working/FreeC/pilot/working/results/S01.pileup.gz_control.cpn
..will remove all windows with read count in the control less than 50
Initial guess for polynomial:
Error: variation in read count per window is too small.
Unable to proceed..

Thanks a lot!!!
Yihua

[ffavero]

Apologies if it's unrelated, but since nobody replied yet I just post it:
I see there is a "sequenza" mentioned in you file path.
Didn't sequenza worked out for the exome comparison with control?

[morrowliu]

Hi, ffavero,

Sequenza works out fine for the normal vs. control. But the results from genome wide plot and chromosome-wise plot are inconsistent, which cause me concerned.
So I was thinking of trying FreeC.

Yihua

[ffavero]

Hi Yihua,
what do you mean by inconsistent? Sorry if I drag the thread even more off-topic.
If you have time to describe the problem I might be able to fix the issue in sequenza (if you want).
Best
Francesco

[morrowliu]

Hello Francesco,

Sorry for the late reply, I haven't been working on some other projects and now just went back and check what I found the "inconsistence" issue in Sequenza. FYI, I didn't carefully go through the paper so I don't know if I misread some part. If I did, it will be really good that you can point it out and help me with Sequenza.

So I ran those recommended processes in Sequenza user guide. But I did two plots, one is the "chromosome.view" function, another is the "sequenza.results" function. I assume the genome wide CN calls from both functions should be very similar. However, when I ran the my mouse WES data sample, I found that the CNV calls from these two functions are different.

Hope you can provide me some insight about better using Sequenza! I really like it is step-by-step user guide!

Thanks!

Yihua