Minimally-invasive sampling is commonly used to obtain samples from rare, elusive or dangerous animals. However, this sampling technique often results in samples that are too low in quality or quantity for successful use with most high-throughput sequencing methods. Using cloacal swabs from the threatened Western Rattlesnake (Crotalus oreganus), Danielle Schmidt and colleagues show that Genotyping-in-Thousands by sequencing (GT-seq) can successfully be used to generate high-throughput sequence data from low-quality, low-quantity samples. We interviewed Danielle Schmidt (first author) and Professor Michael Russello (last author) to find out more about what went on behind-the-scenes of this study.

What led to your interest in this topic / what was the motivation for this study? 

Conservation genomics has become an increasingly common term in the literature, yet many study systems that involve elusive or at-risk species must rely on minimally- or non-invasive sampling to meet research and management objectives. Although a valuable source of biological material, DNA extracted from minimally- or non-invasive samples is typically of low quantity, poor quality, and contaminated with exogenous DNA, all of which may be incompatible with modern sequencing technologies. Implementing leading-edge genetic and genomic tools to study conservation-related questions has been a long-standing interest in the Russello Lab.

What difficulties did you run into along the way?

Based on earlier work that came out of our lab (Russello et al. 2015 PeerJ), we suspected that employing a non-targeted sequencing approach like RADseq would not be efficient for collecting genotypic data from minimally-invasive samples. Therefore, we decided to test the efficacy of GT-seq (Campbell et al., 2015), as it is a targeted method that could help circumvent the typical issues involved with sequencing and genotyping lower quality DNA. Our biggest challenge was designing a GT-seq SNP panel that minimized ascertainment bias to ensure our downstream estimates of within- and among-population variation would be accurate. Also, given the number of samples and loci we planned to analyze simultaneously, optimizing the workflow for data collection took some time.

What is the biggest or most surprising finding from this study? 

One of the most surprising findings was the exceptionally high genotype consistency between paired blood and cloacal swab samples genotyped with GT-seq, and those blood samples genotyped with both RADseq and GT-seq. We even found that samples with initial concentrations as low as ~0.5 ng/uL successfully amplified, which is promising for future applications of GT-seq with minimally- and non-invasive DNA samples.

Moving forward, what are the next steps for this research? 

We are now exploring the application of GT-seq on a host of species to provide rapid, cost-effective genetic information to support research in molecular ecology and to assist wildlife and fisheries management. We are also testing the performance of this workflow with other non-invasive sample types, including feces and hair. Moving forward, we will be exploring ways of deploying these tools in the field to inform management decisions in real-time.

What would your message be for students about to start their first research projects in this topic?

An important message we would like to convey is to think carefully about potential biases when designing a panel of markers to target, as the composition of your panel must be tailored to your research questions. For example, some applications of GT-seq may seek to intentionally maximize the among-population component of genetic variation in order to identify individuals of unknown origin to a particular fish stock with high confidence. In other cases, as with our study, we wanted a panel that could be used to most accurately reconstruct population structure and connectivity, which we were able to subsequently validate relative to a larger RADseq dataset.

What have you learned about science over the course of this project? 

This project highlighted the benefits of taking a new approach to address a long-standing challenge. In molecular ecology and conservation genetic studies, minimally-invasive sampling is commonly employed as either a required or a preferential approach for obtaining sufficient sample sizes. Yet, it has been recognized since the advent of non-invasive genetic sampling in the 1990’s that issues associated with DNA quality and quantity require careful consideration and extra quality control steps. Today, these considerations also apply to the use of modern DNA sequencing technologies from suboptimal starting material; however, GT-seq provides a versatile approach for overcoming DNA quality issues and providing the population-level data needed to address research and management objectives.

Describe the significance of this research for the general scientific community in one sentence.

Multiplexed, amplicon DNA sequencing, such as that employed in GT-seq, is compatible with the minimally-invasive sampling often required for obtaining population-level data to inform biodiversity conservation.

Describe the significance of this research for your scientific community in one sentence.

GT‐seq offers an effective approach for genotyping minimally-invasive samples, providing accurate and precise estimates of within‐ and among‐population diversity metrics relative to genome-wide approaches such as RAD-seq.

Read the full study here:
Schmidt, Danielle A., et al. “Genotyping‐in‐Thousands by sequencing (GT‐seq) panel development and application to minimally invasive DNA samples to support studies in molecular ecology.” Molecular ecology resources (2020). https://doi.org/10.1111/1755-0998.13090