Investigating bacterial load and profile in sequential tissue cryosections
Hi Michael,
If you are doing amplicon metagenomics, then in my opinion, there is no minimum amount of DNA, you just want to amplify the DNA. The recommend amount by earth microbiome project is 5ng and I think it's the same in Illumina protocol. However, I have amplified DNA that was 2ng/ul and it went fine. In case you were asking about the amount of DNA after indexing and normalization but before denaturation is 4nM. Illumina suggests to start your first run using a 4 pM loading concentration and adjust subsequent runs appropriately.
Please let me know if you have other questions
Good luck,
Ahmed
Source:
http://web.uri.edu/gsc/files/16s-metagenomic-library-prep-guide-15044223-b.pdf
Hi Ahmed, thank you for the response. I intend doing the shot gun if i have enough bacterial DNA. However, if not then i try the amplicon method.
I would consider the question you are asking here. If you are interested in abundance, then amplification may not be the best way to go due to the attributed bias. In which case I would consider a library preparation in which you can reduce or remove PCR as much as possible e.g. KAPA hyper prep. For this you would need at least 250ng total, usually easily achieved in tissue sections. If you are not concerned about abundance and your yield is low, 1 ng is recommended for Nextera, although I have managed with 0.5 ng input.
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