Hey everyone,
my question is maybe strange at first glance, but simple: is the rapid 16S kit's only real advantage the significantly larger 16S data amount generation? Shouldn't I be perfectly able to collect necessary strain-level diversity 16S data on the data analysis level from a total nanopore metagenome, without the PCR bias, given enough sample input? If the above thinking is correct, would you consider triple-digit ng input (below 1ug) sufficient, at least for key players of a mixed microbial community?
Just trying to understand if I really need the 16S barcoding kit since I have the native one (which I will use for total metagenome anyway)
Cheers
A
Sure you can do both, but for the calculation of relative quantifications and for pooling many samples, PCR and barcoding is important.
How would you pool lets say 200 samples with native kit?
Abhijeet Singh both kits offer the same multiplexing capacity, if I understand the question you're asking - both 16S kit and the native kit that we have are "24 barcoding", native / 16S.
I am rather curious about the necessity of 16S in terms of sequencing success - I can see low complexity microbial samples getting sequenced just as succcessfully with a native kit as with 16S, but without the PCR amplification bias, which in fact affects relative quantification negatively, rather than being prerequisite for it as you seem to state (becasue amplification efficiency drops steeply after 60%+ GC content of the amplicon). PCR amplification probably makes a positive difference when trying to detect low-abundance species, but I am not interested in those in this project.
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