Does presence of samples with high microbial load affect samples with low microbial load in a 16S run?

Hi! I am trying to compare and contrast between samples that usually have very high microbial load with those which have very little. Does the presence of such highly different numbers between samples affect the quality and taxonomic depth at which we get information from the low bacterial count samples? Thank you!

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Hello :wave:

I'm not an expert on sequencing, but hopefully I can be helpful.

Sounds like this is your question:
microbial load ->?-> reads per sample

I would add some more detail about what's between microbial load and reads per sample:

microbial load ->
  extracted nucleotide concentration (ng/ml) ->
    PCR products concentration (ng/ml) ->
      reads per sample

At a lab I used to work at, the person in charge of sequencing greatly recommended normalizing the mass of PCR product to a consistent number of grams (or moles) for each sample in order to get more even reads per sample.

This seems to reduce differences in sequencing depth from ~100x variance to ~10x variance, which is a pretty good improvement.

Without this normalization step, more microbes means more DNA means more PCR products means more reads... :boom:

So yes, more samples with high microbial load can overwhelm samples with less.