INTRO
“How many angels can dance on the head of a pin?” This question has been asked many times throughout history as either a serious question about some metaphysical or spiritual topic or as a facetious, rhetorical question meant to exemplify an unanswerable and pointless query. Either way, a much more relevant question to microbiome research is how many microbes can dance on the head of a pin. To a microbe, that pinhead probably looks a lot more like the narrow peak of a very tall mountain, and is likely to start getting crowded with only a few other microbes joining the party. In order to analyze this microbiome, specialized methods are needed to get an accurate picture of the collected sample while avoiding the introduction of any significant biases to the analysis.
THE CHALLENGE
While PCR-based microbe detection methods require very low input DNA, they are often only capable of identifying and/or quantifying a limited number of microbial targets. NGS-based methods are much more effective for picking apart the diversity of a microbial sample, but at the expense of historically requiring a much larger input of DNA, a requirement that is not always easy to fulfill.
A standard approach to this challenge is some form of whole genome amplification (WGA), but WGA often presents a risk of introducing significant biases that can cause the final result of the sequencing to be an inaccurate representation of the genome. This often occurs because WGA methods are susceptible to preferentially amplifying some genomic sequences while avoiding others (often based upon factors such as the GC-content of the region in question).
THE SOLUTION
Zymo Microbiomics Services was able to recover an accurate picture of a microbial standard down to the 100 femtogram input level using our in-house method for low-input shotgun metagenomic sequencing. This method produced little to no discernible bias or distortion of the expected makeup of the mock microbial community and worked well across gram-negative, gram-positive, and fungal microbes.
For a more practical, real-world example, we examined skin swab samples. Skin swab samples are notorious for having extremely low bacterial biomass when collected and will often strain extraction and library prep methods to their breaking point. Using our in-house method, we were able to not only identify and characterize the C. acnes microbial population present in the swab sample, but also identify and characterize several much more uncommon and lower-abundance potential skin pathogens both bacterial and eukaryotic.
CONCLUSION
While many different sample types have worked quite well for shotgun metagenomics sequencing without presenting major challenges, low-input samples are inherently difficult and present unique challenges during the library preparation and sequencing portion of a microbiome analysis pipeline. The Zymo Microbiome Services pipeline for handling low-microbial input samples can successfully process even samples in the hundreds of femtograms or lower without producing significant distortion of the sample’s nature. Please reach out to Microbiome Service Specialist if you would like to know more about how we might be able to process your most challenging microbial samples.