Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR by Boryana Doyle & Gabriella Z. M. Reynolds & Mai Dvorak & Dylan G. Maghini & Aravind Natarajan & Ami S. Bhatt instant download

Absolute quantification of prokaryotes in the microbiome by 16S rRNA qPCR or ddPCR by Boryana Doyle & Gabriella Z. M. Reynolds & Mai Dvorak & Dylan G. Maghini & Aravind Natarajan & Ami S. Bhatt instant download

Measurements of prokaryotic absolute abundance can provide important • This protocol quantifies prokaryotic concentration in stool insights into human gut microbiome biology and correct misinterpretations samples by measuring 16S rRNA of relative abundance data. Despite the existence of several relatively wellgene concentration with qPCR or ddPCR and correcting for stool established methods for making these measurements, most microbiome sample moisture content.studies do not report absolute abundance. To enable researchers equipped • Absolute prokaryotic with standard molecular biology capabilities to incorporate absolute quantification can provide further quantifcation into their microbiome studies, we present a detailed, step-byinsights into microbiome biology step protocol for rigorous and reproducible quantifcation of prokaryotic and correct misinterpretations of the relative abundance data concentration in stool samples. We include methods for measuring stool most commonly reported sample moisture content, quantifying the concentration of the 16S rRNA in microbiome studies.prokaryotic marker gene by qPCR or digital droplet PCR (ddPCR) and analyzing the resulting data. We also highlight and provide strategies to Key referenceovercome common pitfalls of the quantifcation method, such as 16S rRNA gene contamination. The fnal output of this approach is 16S rRNA copies per Maghini, D. G. et al. Nat. Biotechnol. wet or dry gram of stool. In cases where samples have matched metagenomic 42, 328–338 (2024): information, data can be converted into absolute concentration of prokaryotes and taxon-specifc absolute concentrations. To enable researchers to choose the appropriate method for their specifc applications, we also compare and contrast our qPCR and ddPCR methods. In 4 days, ~80 samples can be taken from frozen stool to absolute concentration by using qPCR or ddPCR without the need for resequencing. Overall, this pr