Cultivation-independent microbiome research frequently requires the study of microbial DNA via its isolation and sequencing. However, microbes can vary by size, shape and morphology making their DNA easier or more difficult to isolate. This can lead to differences in microbiome results, depending on the extraction technique used. This blog will outline the basics of DNA extraction, the effects of different DNA extraction techniques on microbiome results, and the steps you can take during DNA extraction to ensure unbiased and consistent results.
The basic steps of DNA extraction
Extraction of DNA from any type of sample relies on three basic steps:
- Lysis: Lysis refers to the process of breaking open cells in order to release their contents, including DNA. This is usually conducted by chemical processes (adding enzymes) and mechanical processes (shaking/mixing).
- Precipitation: Once broken open, it is necessary to separate the DNA from all of the other cell contents. This is done by adding a salt solution and alcohol.
- Purification: The isolated DNA is then washed to remove other impurities and then resuspended in a water solution.
Whether it is a stool sample, skin swab or soil sample, or whether you are using a commercial extraction kit or a classic extraction from scratch, all DNA extraction processes will encompass these three steps. However the exact techniques to conduct these steps can vary depending on the sample, lab, extraction kit or protocol.
Microbiome results depend on extraction technique
Microbiomes are, by default, mixtures of many different types of microorganisms, which differ in shape, size and structure. Therefore, some of these microbes will be easier to extract DNA from, whilst others are more robust. This can cause biases in results in microbiome research studies, whereby certain microbes appear overrepresented and others underrepresented due to their DNA extraction efficiency.
Your microbiome results can be affected by the type of DNA extraction method that you use. Gut microbiome studies have found differences in results from different commercial DNA extraction kits and large quality control microbiome studies have found that DNA extraction is responsible for a large proportion of variation in microbiome profiles. These differences have also been found beyond the gut microbiome, meaning that DNA extraction methods can impact the results of urinary microbiome, skin microbiome and environmental microbiome studies, amongst others. Fortunately, biological variation tends to outweigh the technical variation caused by different DNA extraction kits. However, it is always important to consider the impact of DNA extraction on microbiome results and aim to minimise its impact.
Ensuring accurate microbiome representation
There is no single best DNA extraction protocol for all microbiome studies. The best protocol for your microbiome study will depend on your study and sample type. For example, stool and soil samples contain a huge amount of microbial DNA and therefore will be easier to work with. Low biomass samples, such as skin swabs, on the other hand contain much less microbial DNA and higher percentage of host DNA therefore may require different protocols. It is therefore important to optimise your DNA extraction protocol before using it on all samples in your study. There are a number of steps that can be used in your protocol to help with your extraction if you are having issues:
Bead-beating
One of the most common problems encountered in DNA extraction protocols is inefficient lysis. Some microbes are more robust than others and therefore are harder to break open during the lysis steps of a protocol. If these microbes are not lysed effectively, they will be underrepresented in your microbiome data, leading to biased results. Bead-beating involves adding sterilised micro-beads to your sample and adding them to a sample disruptor device in order to mechanically break open the microbial in your sample. This is a useful way to increase lysis efficiency. It is important to note that the size and shape of the beads that you use, can influence your results. It is also important to remember that excessive bead-beating may lead to overrepresentation of particular species or degrade your DNA, leading to issues in downstream sequencing.
Heating
Heating is another technique that can help with samples that are difficult to lyse. Some commercial DNA extraction kits for stool samples, for example, suggest adding a heating step during lysis to help with samples that are proving difficult to extract DNA from. This heating step can be added into most DNA extraction protocols to help improve lysis from particular samples.
Enzymes
Certain sample types benefit from the addition of enzymes during the extraction process in order to assist with DNA isolation. Breast-milk samples and vaginal swabs, for example, can often by difficult to obtain DNA from, and numerous research studies have reported the benefits of adding enzymes such as lysozyme and mutanolysin to assist with DNA extraction. Similarly, the addition of particular enzymes to soil samples can assist with extracting metagenomic DNA from soil.
Cleaning-up DNA
It is not uncommon for your DNA to have low concentration or poor quality after you have extracted it (as measured by 260/280 values of 260/230 values). If you have enough sample to repeat the extraction, then it may be possible to repeat the process and tweak your procedure. However, often precious samples are low in quantity and do not have enough mass/volume to re-extract. ‘Cleaning’ up DNA after it has been extract can help to remove impurities and improve DNA quality as well as increase its concentration. This can be conducted using commercial DNA purification kits or by repeating the precipitation and purification steps in your protocol.
Remove host DNA
Some human microbiome sample types, including skin, oral and vaginal swabs, contain lots of human DNA, which can drown out microbial DNA in your sequencing results. For sample types such as these, commercial kits are available that can deplete human/host DNA in order to maximise the data that you receive from microbial DNA.
Positive and negative controls
Despite their common use in most other research fields, positive and negative controls are too often forgotten about in microbiome analyses. Not only do positive and negative controls help to validate your results, they also can help increase the efficiency of your microbiome research protocols. Negative controls can be included from the point of sample collection (and are particularly important in studies of low biomass samples such as skin or oral swabs) or from the point of DNA extraction, to ensure that no additional microbes make their way into your samples and your results. Positive controls can be purchased commercially. These standardised mixes of different microbes can help to validate whether your extraction procedure is efficiently isolating DNA from a wide range of microbes, from a sample with a known composition and concentration.
Standardizing DNA extraction
Microbiome research is a relatively new field, which means that the techniques used to study microbiomes, including DNA extraction, can differ from lab to lab. In recent years, scientists around the world have attempted to standardize the steps used to conduct microbiome studies, including the DNA extraction step. These studies show that, across different international labs and scientists, standardized DNA extraction procedures can limit the technical variation in microbiome results caused in the lab. Although these standardization studies have largely focussed on gut microbiome research, other large microbiome research projects including environmental samples have helped develop standardized approaches for DNA extraction. Ultimately these microbiome standards will help labs across the world to compare microbiome results more robustly.
No matter which DNA extraction protocol you use, the most important thing to consider is consistency. No procedure is perfect and each procedure will differ based on the study and sample type, however a consistent DNA extraction protocol for all of your samples will ensure that your results are as robust as possible.
Conducting your microbiome study with Microbiome Insights
DNA extraction is the first step for microbiome analyses and can have important effects on your results. At Microbiome Insights, we are experts in conducting microbiome analysis starting from study design through to DNA extraction, sequencing, and bioinformatics. All samples are processed in our CAP-accredited laboratory using validated protocols. If you are looking for help with your microbiome study, read our guide to conducting a microbiome research study or reach out to our team of experts who will be more than happy to help.
Contact us for more information or request a quote for your study!
