How to distinguish Donor-specific Cell-free DNA from recipient Cell-free DNA when we have VCF file after performing next-generation sequencing.
We are trying to detect the donor specific cell free dna in recipient plasma to predict the on going graft rejection. Amount of donor specific cell free dna increases in recipient plasma during the rejection episode/graft injury.
Its a really hard thing to do, and I don't know if it would be even possible with the current methods.
Unless the donor DNA is tagged or labelled for marker based detection or both donor and recipient are genotyped so that the DNA sequences can be compared, it can not be done. For the molecular point of view, both DNA is the same so next to impossible to identify. On sequence level, they need to be compared and for that we need to know about what we are comparing. You see, its getting complicated.
But I would like to see what others have to say about this question.
Hi,
There is one possible method of distinguishing dcsfDNA and rscfDNA. So,after a transplantation the dcsfDNA can be measured with a probe free droplet digital PCR.
You may try evaluating it's utility by separating the recipients into groups as determining the genotype of donor is a bit difficult and not available.
Then quantification of this dscfDNA would show the presence in urine or blood in recipient.
Thus the dscfDNA would fall within a couple of weeks and this may also vary according to the transplanted grafts!
Elevation shows the rejection due to allograft injury, infection etc.
Yet another method is sequence based diagnosis of rejection using Genome transplant dynamics( GTD).
Aishwarya Amirdharaj As per my understanding, both methods you mentioned need both type of DNA to be genotyped for markers, SNPs or barcoded, but the question does not mention anything about it. So in that case, how would you use these techniques in such case, please enlighten.
Actually VCF file would contain variations of a sample to that of the reference sample unless you have information about reference sequence.
These sequences can be annotated with other databases to determine the variations in HLA gene complexes.
As just interpreting from VCF is difficult you may try to annotate and analyse.
To extract data from VCF file you may try with the scikIt-allele provided in python package that helps in analysis of variations in genetic datas.
Also you can try with opening this file in Excel and it would contain different columns For variant details chromosome,RS ID, reference base,alternate bases , quality and filter note Etc.from this the last column called S1 gives the variant frequency,depth/genotype for each variant.
But without such filtering out strategies and sequences I think this is a complex task to deal.
I am sharing some of the possibilities for your work but simple interpretation is a bit tough, it would be a long process.
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