In many dietary DNA metabarcoding studies, sampling and replication tends to be framed around predefined groups:

• Species A vs. Species B
• Dry season vs. wet season
• Treatment vs. control
• Population 1 vs. Population 2

We are taught to ask ourselves: How many samples do we need to collect per group for a statistically robust sampling design?

But what if group identity does not need to be the primary unit of analysis in the first place?

Recent analytical approaches — including the use of unsupervised and minimally supervised machine learning tools — allow ecological patterns to emerge directly from dietary data without requiring us to impose a priori sampling categories on the "groups' that we have under study. When that happens, the logic of replication changes.

Replication still matters.
But why it matters is different.

Designing a dietary DNA metabarcoding study often begins with a deceptively simple question: How many samples do I really need to collect?

There is not a universally “correct” number. We all want to have a large enough sample size for a powerful analysis. But it can be extremely challenging to collect fresh scat samples from wild animals—especially when they are rare and widespread—and then we face the cost of analyzing what we get.

To answer this question, we need to focus mostly on the ecological inferences we want to make. Are we trying to compare groups? Estimate niche breadth? Detect rare food items? Describe seasonal shifts? The number of samples required to detect differences between sample sets is often very different from the number needed to perfectly catalog everything in a diet. So, I want to share some helpful rules of thumb based on experience across a wide variety of study systems...

We have posted detailed new protocols describing our methods to sequence key mammalian DNA barcodes. They can be found together with a growing number of field and lab protocols on the Kartzinel Lab's centralized protocol page.

You will find protocols for both the D-loop of the mitochondrial control region and the 16S marker are useful for identifying a diversity of mammals, and can be routinely amplified from degraded material such as fecal DNA. We have frequently used these protocols to confirm the identity of mammals in studies involving dietary DNA metabarcoding and/or host-microbiome interactions. They are also very useful for phylogenetic analyses. We have used various polymerases over the years, so these protocols may depart slightly from previously published versions (e.g., Kartzinel et al. 2019 PNAS). However, they reflect our current state-of-the-art strategy for routine work and should be generally more cost or time effective as a result of the changes. 

Since June 2025, we have increasingly made our internal lab methods publicly visible on the "Protocols" section of our webpage. We began with some of the most frequently requested protocols that speak to the unique strengths of our lab's work and experience, featuring field-to-lab protocols for collecting and banking dietary samples, parasite samples, and plant barcode samples. We have expanded to include...