Quantitative microbiome profiling in lumenal and tissue samples with broad coverage and dynamic range via a single-step 16S rRNA gene DNA copy quantification and amplicon barcoding

Abstract

Current methods for detecting, accurately quantifying, and profiling complex microbial communities based on the microbial 16S rRNA marker genes are limited by a number of factors, including inconsistent extraction of microbial nucleic acids, amplification interference from contaminants and host DNA, different coverage of PCR primers utilized for quantification and sequencing, and potentially biases in PCR amplification rates among microbial taxa during amplicon barcoding. Here, we describe a single-step method that enables the quantification of microbial 16S rRNA gene DNA copies with wide dynamic range and broad microbial diversity, and simultaneous amplicon barcoding for quantitative 16S rRNA gene amplicon profiling of microbiota. The method is suitable for a variety of sample types and is robust in samples with low microbial abundance, including samples containing high levels of host mammalian DNA, as is common in human clinical samples. We demonstrate that our modification to the Earth Microbiome Project (EMP) V4 16S rRNA gene primers expands their microbial coverage while dramatically reducing non-specific mammalian mitochondrial DNA amplification, thus achieving wide dynamic range in microbial quantification and broad coverage for capturing high microbial diversity in samples with or without high host DNA background. The approach relies only on broadly available hardware (real-time PCR instruments) and standard reagents utilized for conventional 16S rRNA gene amplicon library preparation both of which make it amenable for immediate and widespread adoption. Simultaneous 16S rRNA gene DNA copy quantification and amplicon barcoding for multiplexed next-generation sequencing from the same analyzed sample, performed in a combined workflow, reduces the amount of sample needed and reduces time and reagent costs. Additionally, we demonstrate that using our modified 16S rRNA gene primers in a digital PCR (dPCR) format enables precise and exact microbial quantification in samples with very high host DNA background levels without the need for quantification standards. Potential future applications of this approach include: (1) quantitative microbiome profiling in human and animal microbiome research; (2) detection of monoinfections and profiling of polymicrobial infections in tissues, stool, and bodily fluids in human and veterinary medicine; (3) environmental sample analyses (e.g., soil and water); and (4) broad-coverage detection of microbial food contamination in products high in mammalian DNA, such as meat products. We predict that utilization of this approach primarily for quantitative microbiome profiling will be invaluable to microbiome studies, which have historically been limited to analysis of relative abundances of microbes.

Self-reinoculation with fecal flora changes microbiota density and composition leading to an altered bile-acid profile in the mouse small intestine

“…To answer our second question (Does self-reinoculation with fecal microbiota impact upper GIT microbial composition? ), we performed quantitative 16S rRNA gene amplicon sequencing [38,39] (Barlow JT, Bogatyrev SR, Ismagilov RF: A quantitative sequencing framework for absolute abundance measurements of mucosal and lumenal microbial communities, submitted) on the stomach (STM), jejunum (SI2), and cecum (CEC) samples. Qualitative sequencing revealed dramatic overall changes in the upper GIT microbiota caused by selfreinoculation ( Fig.…”

A Quantitative Sequencing Framework for Absolute Abundance Measurements of Mucosal and Lumenal Microbial Communities

“…Additionally, the total microbial load measurement will be affected by the 16S rRNA primer set chosen and its respective taxonomic coverage. The primers in this study were chosen to have broad coverage and also to limit amplification of host mitochondrial DNA, [31][32][33] to ensure proper quantification of mucosal and small-intestine samples with high host DNA loads. Finally, to take full advantage of the power of this quantitative framework, study designs must incorporate proper sampling techniques to address spatiotemporal variation in microbial abundances.…”

“…To minimize and quantify bias resulting from potentially uneven amplification of microbial 16S rRNA gene DNA, or non-specific amplification of host DNA, we utilized dPCR in a microfluidic format. [31][32][33] . dPCR is an ultrasensitive method for counting single molecules of DNA or RNA.…”

“…Universal primers to calculate the total 16S rRNA gene concentrations were a modification to the standard 515F-806R primers 4 to reduce host mitochondrial rRNA gene amplification in mucosal and small-intestine samples (Table S6). [31][32][33] Thermocycling for universal primers was performed as follows: 95 °C for 5 min, 40 cycles of 95 °C for 30 s, 52 °C for 30 s, and 68 °C for 30 s, with a dye stabilization step of 4 °C for 5 min and 90 °C for 5 min. All ramp rates were 2 °C per second.…”

“…Extracted DNA was amplified and sequenced using barcoded universal primers and protocol modified to reduce amplification of host DNA [31][32][33]…”