Why DNA concentration and quality is too low from Stool samples of infants?

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Maurice Ekpenyong

I do not know what your definition of 'infant' is. However, I understand that the concentration of DNA in 0-12 month old infant stool samples is a function of quantity and quality of microbiota. Infants fed with breast-milk and those fed with formula have very different levels of microbiota. Infant age is also a factor. Please see the link for details. https://academic.oup.com/femsec/article/54/1/77/566042

Abhijeet Singh

"DNA concentration and quality is too low"

>> low as compared to what?

Ambika Dudhate

Abhijeet Singh : Thank you for the reply, I was considering the fact that to sequence the DNA for metagenomic analysis the good concentration should be 200ng/ul with absorbance at 260/230 >1.8 (my criteria of comparison). So I was thinking whether it will be a good option to carry out the metagenomics with the DNA concentration and quality I am getting or should I a try some alternative methods to isolate the DNA that might increase the concentration in infants (0-4 months) stool samples.

Maurice Ekpenyong : Thank you for your answer and suggestions.

There are few issues with this question:

" DNA sequencing for metagenomic analysis " - this does not say anything what kind of analysis you want to perform on the samples. Amplicon- or whole genome metagenomic sequencing.

Further, 200 ng/µl is exceptionally high concentration for the purpose. I don't know how did you get that number.

Sure DNA quality can be a issue but nanodrop reading are just indication and not to be trusted. For actual concentration qubit is preferred and most of the sequencing facilities, if not all, prefer qubit concentrations. For real quality check you need use bioanalyzer, as done by sequencing facilities.

For sequencing, only few nanograms (3-5 ng) (amount and not concentration) of library is needed, and library prep depends on the type and method used.

If the sequencing is 16S amplicon based, the quality or concentration should be measured for the library. Generally PCR with 16S primers do not have much problems with quality of DNA and your quality is not at all bad.

The quality and amount of DNA would depend on the starting material and the whole extraction process. If you want to increase the concentration, elute the DNA with less volume of elution buffer (instead of 100 µl as kit)

Alexander Sasha Vlassov

we also observed abnormally low amount of DNA purified from some infant stool samples. one solution is to simply process more stool. However another potential problem could be a suboptimal "preservative" - certain solutions claimed to preserve the sample - could be either incompatible with your DNA/RNA purification kit (thus low yield), or might not be actually doing a good job at "stabilizing" the nucleic acid in your sample. We try to process our samples fresh, or keep them frozen at -80C

Alexander Sasha Vlassov : Thank you very much for your feedback, Yes our stool samples are in preservatives and have little biomass in them, may be that is the biggest reason we are getting this low concentration of the DNA.

Alvin Jogasuria

Ambika Dudhate, MP Biomedicals provides extraction kit for this sample type. You'll get better yield and quality. https://www.mpbio.com/us/116570200-fastdna-spin-kit-for-feces

Article Comparison of seven methods for extraction of bacterial DNA ...