Next-generation sequencing is a hugely powerful tool in microbiome science to study what microbes and microbial genes are within a certain environment. This has led to major advances in our understanding of human health, environmental science and basic biology. However, next-generation sequencing generally only tells you the proportion of particular microbes within a certain environment relative to all other microbes. In most cases, it does not quantify the absolute number of micro-organisms in that sample. In this blog, we discuss the importance and approaches for quantification in microbiome studies.

1. Relative versus absolute abundances 

Microbiome science has come a long way in the past 20 years. Previously, the most common approaches to studying microbiomes were to culture bacteria, by growing different species on different types of nutrient agar, or using qPCR, to quantify microbial species in a sample one at a time. The advances in next-generation sequencing technologies, however, allowed microbiome researchers to study thousands of microbial species and strains simultaneously and to identify new species that have not been discovered before or easily cultured in the lab.

Despite its strengths, traditional microbiome sequencing also has some drawbacks. In general, microbiome sequencing results are proportional, meaning that they are usually reported as relative abundances out of 100%. This means that, typically, they do not provide absolute quantities of specific microbes within a sample, as counted by the number of cells or DNA copies. For example, using relative abundances, two people may both have 20% staphylococcus in their skin microbiome, however, if one of these people has double the microbial load on their skin, they will have twice as much staphylococcus as the other, using absolute abundances. The absolute quantity of total microbes or specific microbes within a sample can be an important factor, especially in studies that include samples with low microbial biomass, including skin swabs or air samples. So what are the approaches for quantification in microbiome science?

2. Quantification approaches

There are a variety of ways to obtain absolute quantities of microbes within a sample:

• Culturing: Culturing involves growing bacteria, viruses, fungi or other microbes within your sample on some form of media, usually nutrient agar. This is an effective way of quantifying living microbes within your sample, but requires samples to be collected and stored appropriately so that these microbes stay alive. There are also some limitations to the types of microbes that can be easily grown in the lab. Many bacteria require strict anaerobic conditions, or particular nutrients to grow, which makes it difficult to grow and quantify certain species within your sample.

• qPCR: Quantitative PCR (qPCR), by definition, quantifies the relative abundance of genes within a sample. It can therefore be used to quantify the total microbial load and specific microbes within a sample. By amplifying the 16S amplicon, qPCR can quantify the total bacterial load within samples. This is particularly important for low biomass samples, such as skin swabs or air samples, as it can confirm whether the bacterial load is large enough to provide accurate and meaningful sequencing results. qPCR can also be used to quantify the absolute abundances of specific microbial species or strains within a sample. qPCR is a highly sensitive technique, however, unlike metagenomic sequencing, it is targeted, meaning it only targets one selected gene at a time and also introduces amplification bias, which can potentially influence results.

• Flow cytometry: In recent years, microbiome sequencing has been combined with flow cytometry to quantify sequencing results. Flow cytometry is a technique that can count individual cells and cell types. By combining the two techniques, it is possible to quantify the absolute abundance of different species within a sample. One study used this combination of techniques and found that microbial load in people with a Bacteroides-enterotype microbiome was associated with Crohn’s disease. Flow cytometry is a very useful technique for quantifying individual cells but also requires additional laboratory analysis if combined with sequencing and it also only counts live cells, which can bias microbiome results.
• Internal standards: Adding an internal, spike-in DNA or microbial standard during DNA isolation and prior to sequencing is another method that can help to determine absolute quantities of microbes within a microbiome. DNA spike-ins are being used in more and more microbiome studies to quantify microbial loads but they also add an extra cost to microbiome analysis.

3. Considering when to quantify

Absolute quantification is not used in all microbiome studies due to the additional costs, time and practical limitations involved. However, there are various circumstances where absolute quantification can help with your study.

• Low biomass samples: Samples such as stool and soil are dense in microbes, however samples such as skin swabs, air samples or respiratory microbiome samples all tend to have very low microbial loads. If these samples are being used for sequencing, it is essential to know whether the microbial load is big enough for amplification, which can be assessed using qPCR. In addition to assessing microbial load as a quality control check, the microbial load of low biomass samples may be critical to differences between experimental groups in your study. 

• Studying antibiotics: Antibiotics significantly reduce the microbial load in a host in addition to changing the composition of microbes present. If your study involves the use of antibiotics (for example using antibiotics to deplete the gut microbiota in mice or studying a human population exposed to antibiotics), microbiome quantification can be very useful to determine the effect of antibiotics on overall microbial density and absolute microbiome abundances.

• Longitudinal studies: The microbial load of a microbiome and the absolute quantities of particular microbes within it can change over time as a result of disease, medications, diet etc. However, using relative abundances may mask these changes that occur in microbiomes over time. For example, a longitudinal study of preterm infants’ microbiomes found that using relative abundances masked blooms in Klebsiella and Escherichia that occurred over time in some children. However, absolute quantification showed that these changes were occurring. Therefore, absolute quantities of particular disease-causing microbes, or indeed health-associated microbes, can change over time but not be picked up by typical relative abundance analyses.

Absolute quantification can either act as an important quality control check or can add another dimension to your microbiome analysis and provide deeper insight into your samples. The team at Microbiome Insights has the expertise to help with your microbiome study so if you have any questions about quantification or any other aspect of your study, then get in touch.