New advances in microbial community profiling: A future without bias
June 12, 2018
The foundation for the study of microbial communities are multiplex PCR and 16S and 18S rRNA gene sequencing analysis1. Using high throughput sequencing in the analysis of microbial communities has led to the new scientific field of metagenomics – the analysis of the combined genomes of organisms co-existing in a community2.
By their nature, complex microbiome samples pose challenges to researchers trying to achieve accurate, unbiased microbial community profiling3. According to Dr. Eric Lader, Senior Director, Product Development at QIAGEN. “Avoiding the various sources of bias is imperative for generating meaningful and representative microbiome profiles – Assay sensitivity limits, especially with low biomass samples, inhibitor co-purification during cell lysis and extraction and contaminant microbial DNA in PCR reagents are major sources of external profiling bias. Low sequence diversity and high GC content inherent to many microorganisms make primer design difficult and can lead to significant amplification bias.”
Imagine a future with no bias
The scientists at QIAGEN have developed the UCP Multiplex PCR kit to help you get meaningful, unbiased results from your complex microbiome samples. Designed for the challenge of microbial samples, the kit features inhibitor-resistant amplification – even with templates with up to 70% GC content. The Ultra-Clean Reagents in our UCP Multiplex PCR Kit, are nucleic acid depleted and tested for bacterial and fungal DNA contamination.
Our R&D data below, shows how the UCP Multiplex PCR Kit can give you the most accurate microbiome profiles.
Get superior sensitivity with no background signal
The UCP Multiplex PCR kit gives unsurpassed sensitivity and specificity, amplifying even very small input amounts with no background signal.
Figure 1. 16S sequences from 20, 5 and 2 E. coli genome equivalents were amplified. With the UCP Multiplex PCR kit small input amounts without background could have been reliably detected while competitor kits were unable to distinguish small template amounts from NTC (Non- Template Control) backgrounds. Amplify templates with up to 70% GC with no protocol or buffer optimization
The UCP Multiplex PCR Kit prevents amplification bias due to GC content, with no protocol optimization required.
Figure 2. Templates with GC contents from 70% to 30% were reliably amplified using the same standard protocol and buffer conditions. Achieve ultra-sensitive detection with no background – in bacterial and fungal samples
Ideally suited to low biomass samples, the UCP Multiplex kit detects as little as 2 E.coli and 3 C.albicans genomes, with no background signal.
Figure 3. PCR reactions of 38 cycles with a total run time of 65 minutes were run. Analysis was performed using the QIAxcel electrophoretic system. As little as 2 E.coli genomes and 3 C.albicans genomes were detected with no amplification in the NTC reactions. The UCP Multiplex PCR Kit has the robustness and specificity required to give meaningful, unbiased microbial community profiles from even your most challenging samples. The UCP Multiplex PCR Kit is available as a stand-alone product and is also a component of the new QIAseq 16S/ITS Panels.
References
1. Sinclair et al. Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform. PLoS One. 2015; 10(2)
2. Petrosino et al. Metagenomic Pyrosequencing and Microbial Identification. Clin Chem. 2009 May; 55(5): 856–866
3. Micah Hamady and Rob Knight. Microbial community profiling for human microbiome projects: Tools, techniques, and challenges. Genome Res. 2009 Jul; 19(7): 1141–1152
