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

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.

Cigarette Smoking-Associated Isoform Switching and 3-prime UTR Lengthening Via Alternative Polyadenylation

Cigarette smoking accounts for approximately one in five deaths in the United States. Previous genomic studies have primarily focused on gene level differential expression to identify related molecular signatures and pathways, but the genome-wide effects of smoking on alternative isoform regulation and posttranscriptional modulation have not yet been described. We conducted RNA sequencing (RNA-seq) in whole-blood samples of 454 current and 767 former smokers in COPDGene Study. We assessed the association of current smoking with differential expression of genes and isoforms and differential usage of isoforms and exons. At 10% FDR, we detected 3,167 differentially expressed genes, 2,014 differentially expressed isoforms, 945 differentially used isoforms and 160 differentially used exons. Genes containing differentially used isoforms were enriched in biological pathways involving GTPase activity and innate immunity. The majority of these genes were not differentially expressed, thus not identifiable from conventional differential gene expression analysis. Isoform switch analysis revealed for the first time widespread 3-prime UTR lengthening associated with cigarette smoking, where current smokers were found to have higher expression and usage of isoforms with markedly longer 3-prime UTRs. The lengthening of 3-prime UTRs appears to be mediated through alternative usage of distal polyadenylation sites, and these extended 3-prime UTR regions are significantly enriched with functional sequence elements including adenylate-uridylate (AU)-rich elements, microRNA and RNA-protein binding sites. Expression quantitative trait locus analyses on differentially used 3-prime UTRs identified 79 known GWAS variants associated with multiple smoking-related human diseases and traits. Smoking elicits widespread transcriptional and posttranscriptional alterations with disease implications. It induces alternative polyadenylation (APA) events resulting in a switch towards the usage of isoforms with strikingly longer 3-prime UTRs in genes related to multiple biological pathways including GTPase activity and innate immunity. The extended 3-prime UTR regions are enriched with functional sequence elements facilitating post-transcriptional regulation of protein expression and mRNA stability. These findings warrant further studies on APA events as potential biomarkers and novel therapeutic targets for smoking-related diseases.

Usage of functional approach to digitalization of criminal procedure

Different approaches to digitalization of criminal procedure are considered; the conclusion about the need for a functional approach based on a system analysis of procedural activity is substantiated; the internal purposes and functions of this activity subject to accounting for the aims of its system digitalization are highlighted; the functional sequence of purposes and stages of digitalization of procedural activity, which could ensure the transition to electronic justice in criminal cases, is highlighted.