How to calculate copies of circulating tumour DNA from sequencing data?

11 November 2017 3 333 Report

NGS data provides information on variant allele frequency which is to be used for calculating copies of total cf-DNA, variant copies of cf-DNA per ml.

I have used the method described in

Annals of Oncology 27: 862–867, 2016

Detection of ubiquitous and heterogeneous mutations in cell-free DNA from patients with early-stage non-small-cell lung cancer

authors in this paper, based on the assumption that there are 3.3 pg DNA per haploid copy of genome, have calculated the total cf-DNA, variant copies of cf-DNA per ml.

Is there any other method available for calculating copies of ct-DNA?

Nandu Surendran

Hello Sunil,

Would you mind checking out these links? It may help with your question.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4659935/

https://www.nature.com/articles/s41523-017-0028-4

https://www.sciencedirect.com/science/article/pii/S1936523315000716

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428730/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747205/

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0140712

Sunil Pachakar

Hello Nandu,

Thanks for your response. I referred the papers for which you have provided the links.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4747205/

above paper has mentioned formula for calculating mutant copies per ml of plasma through DDPCR

Copies/ml of plasma = C*EV/TV/PV.

PV = Volume of plasma used for cfDNA extraction (ml)
EV = Volume in which cfDNA was eluted (μl)
TV = Volume of cfDNA added to the PCR reaction (μl)
C = copies/20μl (data derived from QuantaSoft)

However my study is based on NGS and above formula doesn't help for the calculation purpose.

Recently i come across one publication Article Circulating tumor DNA shows variable clonal response of brea...

If you could help me to understand the calculation part in this particular publication will solve my problem.

The same query was conveyed to corresponding author of the paper, but there is no response yet from their side.

Sunil Pachakar

Dear Wang,

Thank you for reading and answering my query.

I already have idea about calculating copy number of cf-DNA.

I am looking for calculating variant copies of cf-DNA per ml, which basically gives information on presences of mutated gene in the cf-DNA. I hope my query is more clear to you now. please revert back if you come across such papers.

Thanks once again.

How to increase the yield of cfdna extraction?

Enhanced Yellow Fluorescent Protein (Aequorea victoria) DNA sequence?

Does Napabucasin soluble in dichloromethane and in ethanol?

Could you please recommend some good journal that accept very long revie papers (approximately 6000+ words) in neurosurgery, cancer biology ?

Which cell line should we use to perform biological dosimetry experiments?

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Runs of nucleotides in Sanger sequencing?

Has anyone used the Illumina ITS1 primer sets?

How do you mesh a thin solid in Fluent?

Errors in DNA sequences?