Powering a pilot study: How to design an experimental plan
to compare the performance of collection/preservation methods
The goal of this document is to help researchers generate robust, scalable and reliable data to guide the choiceof an appropriate sample collection method for gut microbiome studies. Although gut-focused, the suggested
study design in this document can be applied to all sample types. A properly designed pilot study will generate
the necessary information to choose an appropriate methodology and will allow you to scale from a pilot to afull-scale research project.
In gut microbiome studies, a fresh stool sample extracted immediately is the ideal scenario. However, thismethod is impractical, especially for large cohorts or field collections; freezing the sample or using a stabilizing
solution are considered the best alternatives. We strongly suggest performing a pilot study to accurately
determine which sample preservation method will perform best in its ability to:•••

Capture an unbiased microbial profile at the time of collection (neutrality
Preserve the microbial profile during shipping and storage (stability)
Rapidly homogenize the collected sample (reproducibility))

To benchmark any preservation method, we recommend the following study design:

Stool sample(n≥10(1))

Fresh unstabilizedCompetitor(Triplicate extractions)(Triplicate extractions)

Extraction
at T=0(Triplicate extractions)

Extraction
at T=x(2)(Triplicate extractions)(Triplicate extractions)(1)

We recommend a minimum of 10 different samples (donors)(2)

x is defined by the elapsed time between the collection (T=0) and sample extraction(ex. It takes into account post-collection storage + transportation + pre-processing storage)

Assessement of reproducibility (), neutrality () and stability ()Figure 1: Recommended pilot study design for comparing different collection and stabilization methods. To make valid comparisons between
preservation methods and avoid introducing extraction bias, we recommend using the same extraction method for all samples. Many suitable
extraction kits are available; our recommendation for optimal lysis, better DNA quality and higher yields is the QIAGEN® QIAamp® PowerFecal®
Pro DNA kit (Evaluation of DNA extraction methods). If you are conducting a study involving mailing of preserved stool, the samples should beexposed to high temperatures, and/or freeze/thaw cycles to simulate real life transport conditions. Samples collected in the field will performdifferently than in a controlled laboratory setting. Ideally, the samples tested in your pilot should be collected from subjects that are representativeof your cohort of interest (matching Bristol scale stool types, adults vs pediatric donors, etc.).

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Sample neutrality and stability
How does the collection method affect the sample’s taxonomic profile?
Neutrality: A frozen or stabilized sample should be indistinguishable from a fresh stool sample that wasextracted immediately. In the suggested experimental design, neutrality is measured by comparing thestabilized samples (at baseline: T=0) to a fresh stool sample that was immediately extracted.
Stability: The microbial profile of a frozen or preserved sample should not change in composition over timeand should be identical to a sample extracted immediately after collection, regardless of shipping and storage
conditions and/or elapsed time between collection and processing. Stability is measured by comparing thepreserved sample extracted at the time of collection (T=0) to the preserved sample extracted post-storage orpost simulated shipping conditions (T=x).
Lack of neutrality and/or stability will result in a sample with a taxonomic profile different from the fresh
sample. This is due to bacterial growth or decay and/or DNA degradation, and results in over/underestimationof select taxa as well as the possible detection of false positives and/or negatives.

Reproducibility
How likely are replicates from the same sample to agree with one another?
Reproducibility: Multiple aliquots extracted from the same specimen should be similar to one another. Stool
can be very heterogeneous, so it is critical to fully homogenize the sample in order to ensure data
reproducibility across multiple extractions. Reproducibility can be tested by performing triplicate extractions
from the same sample (see Figure 1). A wide variety of donors should be included in order to capture thediversity of sample types and their propensity for being homogenized (for example: liquid versus solid stool).
Lack of sample homogeneity can result in a significant taxonomic profile shift due to the lack of reproducibility
from duplicate extractions. This is particularly problematic if your study requires a second extraction to
perform additional testing/experiments.

In Summary
Neutrality, stability and reproducibility are the three most important criteria to take into consideration when
testing or comparing preservation methods. Other important questions you need to ask yourself are: are thequality and yield of the extracted nucleic acids high enough to perform any downstream applications (i.e., NGS,long-read sequencing, etc)? Can the sample be collected in remote locations and be preserved during theshipping process? Have I tested the specific cohort of interest (infants and IBD patients are different from thegeneral adult population)? Does the usability (intuitive and user-friendliness) of the collection method
maximize donor compliance?
It is important to arm yourself with an optimal experimental setup to power the decision making process.
Our suggested experimental design will allow you to compare the right parameters to ensure theoptimal accuracy of each step of your microbiome study.

Technical support is available Monday to Friday (9h00 to 17h00 ET):•••

Toll-free (North America): 1.866.813.6354, option 6
All other countries: +1.613.723.5757, option 6
Email: support@dnagenotek.com

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All DNA Genotek protocols, white papers and application notes, are available in the support section of our website at www.dnagenotek.com.

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