I've been doing some microbiology related work lately, but haven't had too much insight into this, and I knew more about plant ecology before. before, so I'm asking a technical question to you all.
I have recently been given a microbial sequencing dataset based on the 16s method, but I have found that this method does not seem to allow for accurate absolute quantification of microorganisms, what factors affect this? Is it some step in the PCR process? Or is it the high throughput sequencing process? And while exploring this question I learnt about qPCR technology, if we have the right markers, is it possible to get the total amount of microbial in a sample and then get the absolute abundance of the community through relative abundance.
Thanks for any replies!
1) Selecting a hypervariable region is crucial in quantifying bacterial community from 16S rRNA gene copy abundance. No single region can accurately quantify all bacterial phyla present in an environmental sample. The choice of region (e.g., V3 or V9) depends on the specific bacterial phyla being targeted.
For example, if you are interested in quantifying the abundance of Firmicutes and Bacteroidetes in a soil sample using 16S rRNA gene sequencing, you may choose the V4 hypervariable region. However, if your focus shifts to Actinobacteria and Proteobacteria, the V1-V2 region might be more suitable. The choice of hypervariable region depends on the bacterial phyla you want to target for accurate quantification in your environmental sample.
2) Why select a specific region from the 16S rRNA gene? Many sequencing platforms, such as Illumina, lack the capability to sequence the entire length of the 16S rRNA gene (~1500 bp). However, PacBio currently shows promise in overcoming this limitation.
3) Different bacterial species have varying numbers of 16S rRNA gene copies per genome. This can lead to discrepancies in quantification when using 16S rRNA sequencing as a measure of microbial abundance.
For example; There is a variable 16S rRNA gene copy numbers in Escherichia coli and Mycobacterium tuberculosis. Escherichia coli typically has about 7 copies of the 16S rRNA gene per genome, while Mycobacterium tuberculosis has only one copy. If both species are present in a microbial community and are quantified solely based on 16S rRNA sequencing, the higher copy number in E. coli might lead to an overestimation of its abundance compared to M. tuberculosis, even if their actual abundance in the sample is similar.
4) Identifying a single perfect conserved region in the 16S rRNA gene that would enable PCR primer design to capture the entire bacterial diversity is not feasible. The process of PCR amplification (nature of PCR primer), which is commonly used in 16S rRNA sequencing, can introduce biases. Certain bacterial taxa may be preferentially amplified over others, leading to inaccurate quantification.
For example; the 16S rRNA primer set, 515F/806R primer pair, was designed to amplify a broad range of bacterial taxa. However, it has been shown to have biases towards Proteobacteria, Firmicutes, and Bacteroidetes, while underrepresenting others like Actinobacteria and Verrucomicrobia.
5) It is difficult to establish universal standards for calibrating 16S rRNA sequencing data to absolute microbial abundance due to the complexities of microbial communities and variations in DNA extraction, PCR amplification, and sequencing protocols.
For example, one laboratory might employ a method that efficiently lyses certain bacterial cell types, resulting in higher DNA yield, while another laboratory might use a different method that is less effective for lysing those same cells (especially when working with marine sediments). Consequently, variations in DNA extraction efficiency can lead to differences in the amount of DNA extracted from microbial samples, affecting the accuracy and reproducibility of microbial abundance estimates obtained through 16S rRNA sequencing.
Hope this helps.
Chinnamani PrasannaKumar's answer is insightful. However, one should be wary of the effect of copy numbers of the 16s gene during qPCR as well. Be sure to know the copy nos. of the gene if you want to be 100% sure in expressing the absolute numbers of bacteria. Otherwise, interpret it accordingly if the copy nos. is more than one or unknown.
One more point to add to the 16s relative abundance is the choice of bioinformatics pipeline. The choice of pipeline can yield significantly different abundances (or not) but it is bound to make some noticeable changes in the abundance. Hence, we prefer the term 'relative abundance' as it compares the abundance of a bacterium compared to others in a given scenario of DNA extraction, processing, sequencing platform, and the bioinformatics pipeline used. Article A comparison of sequencing platforms and bioinformatics pipe...
Just qPCR is nto reliable because sometimes, different speceis can have 99-100% identical 16Srna. If you want to quatify something specific better for you use CARD-FISH with an specific marker.