Sequencing on the edge: Tools and techniques for sequencing low biomass isolates from unicellular eukaryotic...

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. 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.