What is the typical host-to-bug DNA ratio found in nanopore sequencing without amplification?

Question

I'm interested in sequencing a human sputum sample using an ONT MinION without performing any type of whole genome DNA amplification or targeted PCR.

Has anyone found a good reference (or anecdotal evidence) for a range of human to pathogen DNA ratios typically found in sputum samples?

Does this depend on whether the patient is infected by a virus or bacteria, and the species that is infecting the patient?

I am seeking a ballpark estimate. Can I expect to find 10x as much human DNA as pathogen DNA, or 100x-1000x?

Answer 1

My recollection of a conversation I had with Justin O'Grady is that it's about 99% host DNA for unfiltered samples. There are a few ways to reduce that portion prior to sequencing. He has written a paper that mentions a saponin-based host depletion method that is quite effective:

https://doi.org/10.1038/s41587-019-0156-5

Answer 2

Anecdotally, I have been told that it varies from person to person, also might be affected by disease or hygiene in a particular environment. Here is an article dealing with that problem:

https://journals.plos.org/plosone/article/comments?id=10.1371/journal.pone.0076096

Not sure if they have references useful for you in their paper, but they describe a way of enriching microbial or human DNA. It might be helpful.

Answer 3

The disparity is three logs minimum.

The human genome is massive 2 billion bp, bacteria are 100 million and viruses 10000 bp for RNA viruses. The copy number of the pathogen therefore has to be huge to compensate for the disparity in genome size.

Think about it for a minute if it is 1% then it means for each peripheral mononuclear blood cell (PMBC 'white blood cells') there are 20 million e.g. bacteria ... thats a dying person. The immune system would be incapable of coping. In an infection of that intensity the PMBCs would flood the systemic circulatory system, maintaining a clear imbalance in genome disparity.

You are very unlikely to obtain any DNA viruses abeit they have large genome upto 200Kbp because they are present at low copy number (given smallpox is eradicated).

Where there is a higher chance of success if the individual is pregnant, which causes immunosuppression to protect the foetus or immunocompromised for example HIV, transplantation drugs or chemotherapy amongst others.

The other scenarios you are likely to encounter 'success' involve extremely virulent pathogens such as Ebola (RNA virus), later stages of septicaemia etc..