Quantitative Microbiome Profiling

The development of next-generation sequencing (NGS) has instigated research on the role of the microbiome in health and disease. However, it becomes a challenge to identify those microbial taxa that are truly associated with an intervention or health outcome due to the compositional nature of such microbiome datasets. Quantitative microbiome profiling is a method that overcomes the compositional structure of microbiome sequencing data by integrating absolute quantification of microbial abundances into the NGS data. With the rich experience and excellent experts in gut microbiota, Creative Biolabs launches quantitative microbiome profiling to help you complement 16S rRNA gene sequencing with estimates of microbial load.

Our Service

QMP

• Characteristics:
• Microbial cell counting using flow cytometry
• Complement 16S rRNA gene sequencing with estimates of microbial load by quantifying bacterial cells or 16S rRNA copy number
• Bypass many of the statistical and interpretative challenges that arise from the compositional structure of microbiome sequencing data
• Process:
  Downsizing the samples to an even sampling depth, defined as the ratio between sample size (16S rRNA gene copy-number-corrected sequencing depth) and microbial load.
• Advantages and limitations:
  QMP counts only intact microbial cells. Therefore, new biases could theoretically be introduced when samples contain a significant amount of free extracellular prokaryotic DNA. This free DNA is captured during sequencing but is excluded during flow-cytometry cell counting. In case the taxonomic composition of free circulating DNA differs from the composition of intact microbial cells, this might result in the introduction of a new source of bias in downstream analysis.

QMP-PMA

• Characteristics:
  QMP-PMA is the method that counts microbial cells using flow cytometry combined with propidium monoazide pre-treatment of fecal samples before metagenomics DNA isolation to only profile the microbial composition of intact cells.
• Process:
  Except for the additional PMA pre-treatment before metagenomic DNA isolation, QMP-PMA method is conducted identically to the standard QMP method.

QMP-qPCR

• Characteristics:
• A simple and cost-effective alternative to determine the bacterial load and estimate the absolute taxon abundance from NGS data
• Molecular based quantification of the microbial load using qPCR targeting the 16S rRNA gene
• Process:
  First, the average number of 16S rRNA gene copies per bacterium was calculated for each sample based upon the sequencing data. Second, the total number of 16S rRNA gene copies/gram feces as determined by qPCR was divided by the average 16S rRNA gene copy number of that respective sample. Last, the same approach as for the standard QMP method.
• Advantages and limitations:
• qPCR-based quantification is the cost-effectiveness, simplicity and accessibility.
• Sensitivity is a limitation for QMP-qPCR, as qPCR has been reported to be only sensitive enough to detect 2-fold changes in gene concentration or microbial load.

Fig.1 Microbiome profile comparisons. A) relative microbiome profiling (RMP), B) QMP, C) QMP-PMA, and D) QMP-qPCR. (Galazzo, 2020)

Creative Biolabs provides comprehensive quantitative microbiome profiling services for gut microbiota research. To find out more information about our Gut Microbial Biomarker Discovery Service, please don’t hesitate to contact us.

Reference

1. Galazzo, G.; et al. How to count our microbes? The effect of different quantitative microbiome profiling approaches. Front Cell Infect Microbiol. 2020, 10: 403.

For Research Use Only | Not For Clinical Use