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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

Bogatyrev, Said R. and Ismagilov, Rustem F. (2020) 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. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20200123-094353991

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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.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2020.01.22.914705DOIDiscussion Paper
ORCID:
AuthorORCID
Bogatyrev, Said R.0000-0003-0486-9451
Ismagilov, Rustem F.0000-0002-3680-4399
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. bioRxiv preprint first posted online Jan. 22, 2020. This work was supported in part by the Kenneth Rainin Foundation Innovator Award (2018‐1207), Army Research Office (ARO) Multidisciplinary University Research Initiative (MURI) contract #W911NF‐17‐1‐0402, and the Jacobs Institute for Molecular Engineering for Medicine. We thank Natasha Shelby for contributions to writing and editing this manuscript. Data Availability: NGS data and sequencing data analysis scripts were obtained and are available from [6, 7]. Declaration of Interests: The contents of this article are the subject of a patent application filed by Caltech.
Group:Jacobs Institute for Molecular Engineering for Medicine
Funders:
Funding AgencyGrant Number
Kenneth Rainin Foundation2018‐1207
Army Research Office (ARO)W911NF‐17‐1‐0402
Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Record Number:CaltechAUTHORS:20200123-094353991
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20200123-094353991
Official Citation: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. Said R. Bogatyrev, Rustem F. Ismagilov. bioRxiv 2020.01.22.914705; doi: https://doi.org/10.1101/2020.01.22.914705
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:100868
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:23 Jan 2020 19:01
Last Modified:23 Jan 2020 19:01

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