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

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.

Uncovering bacterial hosts of class 1 integrons in an urban coastal aquatic environment with a single-cell fusion-polymerase chain reaction technology

“…that overcomes these problems by co-amplifying taxonomic markers with functional genes of interest from single uncultured bacterial cells. Single cells are trapped in droplets generated by emulsifying an aqueous phase containing PCR reagents and environmental bacteria in an emulsion oil [21][22][23][24][25][26] . This compartmentalises the fusion PCR into parallel, single cell-level reactions.…”

“…To date, epicPCR has been successfully applied to target functional genes including the bacterial dissimilatory sulfite reductase dsrB gene 21, 24 , the bacteriophage ribonucleotide reductase RNR gene 25 , antibiotic resistance genes 22, 23, 26, 27 , and clinical class 1 integron genes intI1, sul1 , and Δ qacE 23, 27, 28 . In all these studies, epicPCR amplicons comprised short fragments of genes of interest linked to the V4 hypervariable region of 16S rRNA markers.…”

“…epicPCR (emulsion, paired isolation and concatenation PCR), also known as OIL-PCR (onestep isolation and lysis PCR), is a fusion PCR-based technique that overcomes these problems by co-amplifying bacterial taxonomic markers along with functional genes of interest and doing so from single uncultured bacterial cells. Single cells are trapped in droplets generated by emulsifying an aqueous phase suspension of environmental bacteria in an emulsion oil [21][22][23][24][25][26] . This compartmentalises the fusion PCR-type amplification process into parallel, single cell-level reactions.…”

Uncovering Bacterial Hosts of Class 1 Integrons in an Urban Coastal Aquatic Environment with a Single-Cell Fusion-Polymerase Chain Reaction Technology

“…Within each droplet, chimeric amplicons that combine genes of interest with taxonomic markers corresponding to diverse phylogenetic groups are generated for high-throughput sequencing. To date, epicPCR has been successfully applied to target functional genes including dissimilatory sulfite reductase dsrB genes, , antibiotic resistance genes, ,,, class 1 integron associated genes ( intI1 , sul1 , Δ qacE ), ,, and bacteriophage-borne ribonucleotide reductase RNR genes . epicPCR amplicons typically comprise short fragments of genes of interest linked to the V4 hypervariable region of 16S rRNA markers.…”

“…epicPCR (emulsion, paired isolation and concatenation PCR) is a fusion PCR-based technique that overcomes these problems by co-amplifying taxonomic markers with functional genes of interest from single uncultured bacterial cells. Single cells are trapped in droplets generated by emulsifying an aqueous phase containing PCR reagents and environmental bacteria in an emulsion oil. − This compartmentalizes the fusion PCR into parallel, single cell-level reactions. Within each droplet, chimeric amplicons that combine genes of interest with taxonomic markers corresponding to diverse phylogenetic groups are generated for high-throughput sequencing.…”