Sequencing on the edge: tools and techniques for sequencing low biomass isolates from unicellular eukaryotic parasites and other challenging samples

Authors: HSHIEH, KATHERINE - Oak Ridge Institute For Science And Education (ORISE); Maloney, Jenny

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/18/2025
Publication Date: 8/27/2025
Citation: Hshieh, K., Maloney, J.G. 2025. Sequencing on the edge: tools and techniques for sequencing low biomass isolates from unicellular eukaryotic parasites and other challenging samples. Journal of Microbiological Methods. 237. Article e107253. https://doi.org/10.1016/j.mimet.2025.107233.
DOI: https://doi.org/10.1016/j.mimet.2025.107233

Interpretive Summary: Many sequencing protocols require DNA inputs that exceed what is often available from microorganisms that cannot be grown in the lab to increase numbers, single cells, or environmental samples. This limitation makes the genomes of single-celled parasites difficult to obtain and impedes research, because knowing an organism’s complete DNA make up (genome) is key to improving the detection and treatment of important foodborne human parasites. Parasite genome sequencing shares many of the same challenges as sequencing of the DNA from a single cell or a mixed community of cells from an environmental sample such as soil or water. In this review we have compiled and summarized research findings from a variety of studies working with low DNA samples. We highlight that method selection is an important factor influencing experiment success when working with limited quantities of DNA. Based on these observations, we suggest method validation should be considered in experimental design and provide an overview of successful strategies for a variety of sample types. This work benefits scientists from academia, government, and industry by providing resources to assist with method selection and experimental design to enhance genome sequencing from low DNA isolates and improve the data available for understanding and mitigating the risk of foodborne parasite transmission.

Technical Abstract: Low biomass samples are difficult to study as many sequencing protocols require minimum inputs that exceed the DNA available from unculturable microorganisms, single cells, or environmental samples. This limitation makes the genomes of unicellular eukaryotic parasites difficult to obtain as culture methods are unavailable for many species. There are several techniques that can be used to enhance genome sequencing from low biomass samples, including whole genome amplification (WGA). Though WGA helps address some low DNA input challenges, it also comes with contamination and bias issues that can impact sequencing success. There are also WGA free tools and techniques that have been successfully used for low biomass sequencing, some of which require application specific protocol modifications. This review will highlight and summarize techniques that have been applied to a variety of low biomass samples from unculturable microbes to microbial communities. Because unicellular eukaryotic parasite sequencing shares many of the same challenges as other low biomass sample types including single cell and environmental metagenomic sequencing, studies addressing these types of samples are included in this review. We highlight that method validation and selection can be an important factor influencing experiment outcome and success when sequencing low biomass samples, and suggest that when feasible, method validation should be considered in experimental design. Reviewing the benefits and challenges associated with different techniques for enhancing sequencing success for low biomass samples will be useful for those who work within these fields and can assist in method selection and experimental design for exploring challenging samples.