mothur

Experimental Design & Mock Communities

Hi, I am planning to perform amplicon-based sequencing (16s and fungal ITS) of microbiome analysis of multiple samples from a specific niche. I have some experience with the analysis part of the study which follows the sequencing step, as I have done quite a few practice analyses of published sequence data. But my next study involves actual sample collection and handling, which is a first for me. And have a few questions that I hope someone can help me with to be better prepared -

  1. Anyone with experience in this, what are some unexpected hurdles that you had to overcome during your first few studies?
  2. Having a “mock” synthetic community among your samples- Is it necessary? What benefits does it offer? I understand it can help identify errors during the analysis pipeline.
  3. What is the ideal composition of a mock community? Should the microbial compositions be broad and cover all possible branches? Or should it be composed of organisms likely to be found in the niche under study?

Apologies if this is not the right forum for this question, but I could not find a better forum of experienced users regarding this

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  1. Anyone with experience in this, what are some unexpected hurdles that you had to overcome during your first few studies?

A lot could be said on this. Some big points… Have a biological question that motivates what you are doing. Use the V4 region, the two reads need to fully overlap. Be consistent in how you handle your samples, when you sample, etc.

  1. Having a “mock” synthetic community among your samples- Is it necessary? What benefits does it offer? I understand it can help identify errors during the analysis pipeline.

A mock allows you to calculate your sequencing error rate

  1. What is the ideal composition of a mock community? Should the microbial compositions be broad and cover all possible branches? Or should it be composed of organisms likely to be found in the niche under study?

I’d suggest using the ZymoBIOMICS Microbial Community DNA Standard (D6305 or D6306). It will work for most applications. I think it has two fungi in it

Thank you for your reply. I am planning to sample oral microbiome and mycobiome. This may possibly evolve into a study having to culture these oral samples as-such in an artificial environment mimicking the oral environment (microcosm biofilm) followed by sequencing of these artificial polymicrobial microcosm biofilms. I hope you can clarify some further questions I have -

  1. Is there any “trick” to ensure the paired-end reads overlap?
  2. Is the mock community necessary? Can I do a study without it?
  3. Considering some funding limitations, will it be sufficient if I create my own mock community by -
    a. Mixing pure cultures in specific ratio followed by DNA extraction and sequencing of the polymicrobial mixture OR
    b. Extracting DNA from pure cultures of multiple organisms, and then mixing the DNA solution?

Thanks once again!

EDIT: I took some time to go through ZYMOResearch’s Products and I can see 2 products -

  1. Microbial Community Standard (Contains cells which have to be lysed)
  2. Microbial Community DNA Standard (the one you mentioned, contains DNA extracted from their Standard)

So would you recommend using the standard with cells to verify the error/bias in Lysis step too? Or will the DNA standard suffice?