Do We See the Full Microbial Nucleic Acid Picture Without Optimized Sample Prep?
November 7, 2022The last 20 years have seen an implosion in sequencing costs driven by both industrial and academic efforts to bring these sequencing benefits to the individual person. Nucleic acid sequencing has long been touted as an answer for personalized medicine, whether through identifying genetic biomarkers, understanding the proteome through transcriptomics, or unlocking the mysteries of the human microbiome.
With these dramatic cost reductions, it is no wonder that the number of whole genome sequences has increased from roughly 400,000 twenty years ago to the astounding 2,167,900,306 sequences reported by NCBI this October. A red-hot compound annual growth rate of ~53% over the last two decades. During this time, the analytical prowess and technology used to unlock the potential of genomics have changed dramatically. However, many techniques and procedures used to isolate and purify DNA are the same SOPs utilized in laboratories long before sequencing was a ubiquitous term in the life science industry.
For example, the isolation of microbial DNA from stool and soil has long seen homogenization used during the initial sample preparation step to maximize the recovery of target analytes. Researchers have used blenders, vortexes, and various other homogenizers in this first step to ensure the samples are thoroughly mixed. In addition, some researchers utilize bead mills like the Bead Ruptor Elite to mix the samples, lyse the microorganisms, and release the needed intracellular analytes into suspension, where they can then be isolated. Much of the published literature around these methods uses glass beads which have been seen in laboratories worldwide for the better half a century. In conjunction with mixing, these glass beads (in various sizes) have been used to lyse fecal microbes since the 1970s. In more recent years, companies have brought other beads like garnet, carbide, and ceramic media to address the needs of the DNA isolation market.
OMNI's scientists delved into microbial lysis to fully understand what happens inside the tubes during homogenization. They prepared a poster highlighting these findings at the American Society of Microbiology: Microbe conference. In this poster, the experiments were designed around understanding not only the impact of the size of the bead media but also what differences the materials the beads are made from have on lysis.
Observed effects were not limited to a single tested species both gram-positive, and gram-negative bacteria were tested as well as a yeast species.
The data presented in this poster could leave a reader with a very interesting question. Is the sample preparation technique used in their current extraction fully lysing the organisms in their sample?
The answer to that question lies entirely in the lysis step and the use of optimal bead media. If suboptimal bead media is selected, there could be a chance that the complete picture of the microbial community is not being seen. The glass beads cited many times may not be the most optimal media for lysis, especially if the target organisms are gram-positive, this can have dramatic effects on nucleic acid yields, reported population densities, and other metrics relying on the release of intracellular nucleic acids. In almost all cases related to the lysis of microorganisms, our application scientists insist on using 0.1 mm ceramic bead media to optimally lyse target organisms and maximize the recovery of nucleic acids. This bead media compounds both the effect of small and heavy bead media and delivers the most efficient lysis we can offer in a 2 mL tube; with these beads, we see the best results in nearly all microbial applications.
With an optimal selection of bead media, higher yields can be achieved, leading directly to better downstream sequencing data and a clearer picture for the researcher. If efficiency is imperative to you, reach out to one of our homogenizer experts to arrange a complimentary demonstration of our equipment or to have our lab create a custom protocol for your application.
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For research use only. Not for use in diagnostic procedures.