Entering edit mode
4.1 years ago
kaurravneet4123
▴
20
Hi All,
I am confused with the results of CNVkit tool. I am using a flat_reference.cnn against my test sample that has ploidy 4. I am expecting cn = 4 for most of the segments in the .cns file. However, I am getting cn = 2 for most of the segments. Please if someone can explain, why this is the case. Or do I need to change any parameter if the ploidy of the test sample is different from 2? Your quick reply will be much appreciated.
Many thanks, Rav
Ploidy of 2x is assumed by default, so yes, Ploidy of 4 has to be specified or you can divide your final results by corresponding factor.
Dear German,
Many thanks for your reply. Do you mean, I should divide the cn values by 4?
Kind regards,
Rav
Hi,
I think you need to find difference from 4 and then divide by 2 and then plus 2 =)
E.g. CN=6 with ploidy 4
(6 - 4) / 2 + 2 = 3 with ploidy 2. That's what is expected.
Hi German,
Many thanks for your answer.
So my understanding is as below :
if cn=6 while using a flat_reference.cnn and a test sample with ploidy = 4, then
6 - 4 = 2 , this would give you the gene gain or loss for a particular segment of a tetraploid sample.
(6-4)/2, this would give gene gain or loss per diploid genome
then why are we adding 2 in the end?
And is my understanding correct for all the steps?
Sure, the understanding is correct! In the beginning we substracted a baseline - 4 copies. So in the end we have to add baseline - 2, as we assume that the organism is diploid. You may skip adding 2 step - but then you'll have a relatvie change and e.g. for X/Y chromosomes it may be crucial.
I understand now. Many thanks for all the clarification.
As you mentioned earlier that ploidy of 4 can be specified in the pipeline also. Where can I specify the ploidy in the pipeline so that I do not need to do the calculations in the end?
Sadly the option I was thinking about is for different reasons. You need to perform calculations yourself, ie.
https://cnvkit.readthedocs.io/en/stable/pipeline.html
Thank you German for all the above clarifications. Do you think it is appropriate to calculate CNV using a tumour cell line with ploidy=4 against a normal cell line with ploidy=2? If yes, then how can we interpret the copy number from the log2 value?
Basically you have no other choice - you don't have a normal with ploidy of 4 =) copy-numbers have to be corrected for ploidy with the above mentioned procedure - change the baseline from 2 to 4, then calculate differences
Changing the baseline fro 2 to 4 has worked. Thank you so much for all your time and help.
Hi German,
While using a tumour cell line with ploidy=4 against a normal cell line with ploidy=2, if the log2 value of a segment is -0.37681, should I interpret it's copy number as 0.77 or 0.38?
Because log2 = -0.37681 and its antilog would be 0.77.
How should I interpret it's copy number? Sorry, I am bit confused.
hi, log2(CN_tumor / CN_normal) = -0.37681. By default CN_normal = 2. CN_tumor then is equal to 1.549604. And then you need to apply that trick as was discussed before - 4 + (1.55 - 2) * 2 = 3.1. Of course I do not know how CNV-Kit does ploidy correction and these calculations are correct only if CN_tumor is assumed to be 2 by default.
Many thanks for your reply.
I was think the following:
if log2(CN_tumor / CN_normal) = -0.37681
and CN_normal = 2,
then, CN_tumor= (2^-0.37681)*2 = 0.770138593 *2 = 1.540277186
Therefore, CN/diploid = 1.540277186/2 = 0.770138593
Now, if CN_normal = 2, then the CN_tumor/diploid = 0.77
Therefore, if CN_normal = 4, then CN_tumor/diploid should be = 0.77/2 = 0.38
I am more inclined towards CN_tumor/diploid = 0.38 because, if I run the analysis of the same tumor sample with ploidy = 4 against a flat reference, then I get the following result for the same segment:
log2 = -0.387476
Antilog = 0.7645
therefore, CN_tumor/diploid = 0.38225
but CN_normal is not 4. it is 2. your normal sample is diploid. however, I do not deeply understand the design and assumptions of calling, so it is up to you to decide! =)