EpicPCR 2.0 : Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

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Roman , V L , Merlin , C , Virta , M P J & Bellanger , X 2021 , ' EpicPCR 2.0 : Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach ' , Microorganisms , vol. 9 , no. 8 , 1649 . https://doi.org/10.3390/microorganisms9081649

Title: EpicPCR 2.0 : Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach
Author: Roman, Veronica L.; Merlin, Christophe; Virta, Marko P. J.; Bellanger, Xavier
Contributor: University of Helsinki, Department of Microbiology
Date: 2021-08
Language: eng
Number of pages: 15
Belongs to series: Microorganisms
ISSN: 2076-2607
URI: http://hdl.handle.net/10138/334180
Abstract: EpicPCR (Emulsion, Paired Isolation and Concatenation PCR) is a recent single-cell genomic method based on a fusion-PCR allowing us to link a functional sequence of interest to a 16S rRNA gene fragment and use the mass sequencing of the resulting amplicons for taxonomic assignment of the functional sequence-carrying bacteria. Although it is interesting because it presents the highest efficiency for assigning a bacterial host to a marker, epicPCR remains a complex multistage procedure with technical difficulties that may easily impair the approach depth and quality. Here, we described how to adapt epicPCR to new gene targets and environmental matrices while identifying the natural host range of SXT/R391 integrative and conjugative elements in water microbial communities from the Meurthe River (France). We notably show that adding a supplementary PCR step allowed us to increase the amplicon yield and thus the number of reads obtained after sequencing. A comparison of operational taxonomic unit (OTU) identification approaches when using biological and technical replicates demonstrated that, although OTUs can be validated when obtained from three out of three technical replicates, up to now, results obtained from two or three biological replicates give a similar and even a better confidence level in OTU identification, while allowing us to detect poorly represented SXT/R391 hosts in microbial communities.
Subject: PCR-based technic
epicPCR
single-cell metagenomic
taxonomic assignment
high throughput sequencing
ENUMERATION
RESISTANCE
BACTERIA
SEARCH
VIRUS
GENES
11832 Microbiology and virology
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