I am planning to run 16S sequencing on a set of individuals to explore microbial diversity. The question I am facing is: should I sequence them individually or I pool them (for example 5 individuals = 1 sample)? I was just thinking on advantages and disadvantages of both methods:

1) individual. I can get the diversity of each individual, in that way I know the proportion of individuals which have a particular species of bacteria. The cost are higher since I need more samples with respect to the pooled way, but the reads/sample needed should be lower (since this number increases with the microbial diversity).

2) pooled. I have only a general proportion of diversity in the pool (I can't assess how many individuals have a peculiar becterium). The cost are lower due to small amount of samples, but reads/sample should be higher due to more than one individual pooled (?).

Can someone help me? Are my assumptions right or I am missing something?

  • 2
    $\begingroup$ It would help if you shared more about the type of questions you are interested in answering, types of samples you're working with, and sequencing platform. Generally speaking, there's no scientific reason for pooling samples if sequencing them individually is an option. Most next-gen sequencing platforms generate enough reads that pooling is almost never necessary, especially for 16s amplicon sequencing. $\endgroup$
    – MikeyC
    Commented Jun 8, 2020 at 14:35
  • $\begingroup$ Sure, so I have to sequence insects from 5 countries to explore their microbiome. I will use MiSeq sequencing to sequence V3-V4 region. I would like like to know if my thoughts are right and why I should use individual instead of pooled. or viceversa $\endgroup$
    – RP95
    Commented Jun 8, 2020 at 20:20
  • $\begingroup$ By pooling samples one loses a lot of information (it excludes any possible analysis of beta-diversity)... but, yes, it is cheaper. $\endgroup$
    – Roger V.
    Commented Sep 10, 2021 at 7:02

1 Answer 1


Ultimately, it depends on what insights you are trying to glean from your sequencing data, but from a purely scientific perspective, there's almost never a reason to pool samples in the the way you described. You pro-con list is not far off, in terms of information lost or gained, but I think it overlooks some of the down-stream consequences of losing that information on your analysis. For one thing, pooling limits your ability to make biologically or statistically meaningful comparisons. For example, if your individual samples are pooled by country of origin prior to sequencing, you have no way to estimate varation within those pooled samples. Because of that, not much can be said about any observed difference between countries beyond simple descriptions of richness and taxonomy. Treated as individual communities, you can look for differing trends, make meaningful beta-diversity comparisons, and actually analyze them with statistics. If you have environmental data, you might even be able to do some meaningful ecological analyses.

Second, as you mentioned, a pooled sample should theoretically have more diversity than the individual samples it was pooled from (at least it should have no fewer unique sequence variants than the richest of the pooled communities), but pooling could also mask community diversity. If one of the pooled samples has a particularly high abundance community that is dominated by just one or two organisms, it could create the appearance that some organisms at moderate abundance in other more diverse communities are actually vary rare, or non-existant. Even at a much greater depth of sequencing, diversity metrics accounting for abundance lose value, since any outlier sample could skew pooled results, creating a false impression of overall community profile.

Lastly, all of the cost-savings assumptions in your post are going to depend on the sequencing center you use and how they bill samples, so there isn't really a straight answer to give. But I will say that the per-sample cost of Illumina 16s amplicon sequencing is pretty cheap (especially compared to the cost of collecting insects from 5 different countries). And because of how sequencing is performed, pooling samples might not even save you much money. Sequencing is often billed per sequencing kit or per lane of sequencing used, not per individual sample you send. So, unless you have someone to share your sequencing run, it's possible that you'd pay roughly the same amount for 5 samples as you would for 95, with only minimal cost savings on library prep and extraction kits.

To summarize, pooling samples reduces your effective sampling effort and limits your potential scope of inferrence. The only valid reason I can think of for pooling samples in such a way would be if you cannont isolate enough bacterial DNA for sequencing from any one individual (which could be the case, depending on what type of insects you're working with). Like I said at the start, though, it really depends on what you want to learn from your 16s data. If the microbiome comparison is crucial to the outcome of your study, I'd suggest sequencing as many individual communities as possible, or at least a subset of individuals from each country (depending on how many you collected). If you don't plan on doing much analysis beyond describing microbial richness and maybe some taxonomic classifications, maybe pooled samples are sufficient for you needs.

  • $\begingroup$ Thank you so much MikeyC for your very clear answer! $\endgroup$
    – RP95
    Commented Jun 9, 2020 at 6:36

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