Project description:Antibiotic resistance is exacerbated by the exchange of antibiotic resistance genes (ARGs) between microbes from diverse habitats. Plasmids are important ARGs mobile elements and are spread by horizontal gene transfer (HGT). In this study, we demonstrated the presence of multi-resistant plasmids from inhalable particulate matter (PM) and its effect on gene horizontal transfer. Three transferable multi-resistant plasmids were identified from PM in a hospital, using conjugative mating assays and nanopore sequencing. pTAir-3 contained 26 horizontal transfer elements and 10 ARGs. Importantly pTAir-5 harbored carbapenem resistance gene (blaOXA) which shows homology to plasmids from human and pig commensal bacteria, thus indicating that PM is a media for antibiotic resistant plasmid spread. In addition, 125 μg/mL PM2.5 and PM10 significantly increased the conjugative transfer rate by 110% and 30%, respectively, and augmented reactive oxygen species (ROS) levels. Underlying mechanisms were revealed by identifying the upregulated expressional levels of genes related to ROS, SOS, cell membranes, pilus generation, and transposition via genome-wide RNA sequencing. The study highlights the airborne spread of multi-resistant plasmids and the impact of inhalable PM on the horizontal transfer of antibiotic resistance.
Project description:Chromosomes and plasmids are two forms of genetic carriers. Exogenous yeast artificial chromosomes are also considered as yeast centromeric plasmids in many cases. Here, we used state-of-the-art sequencing technologies to comprehensively profile the genetic, epigenetic, transcriptional and proteomic characteristics of an exogenous data-carrying chromosome. We found that the data-carrying DNA formed active chromatin with high chromatin accessibility and H3K4 tri-methylation levels. We also confirmed that the data-carrying chromosome had a circular shape in the nucleus and was arranged in the Rabl configuration, which may contribute to the self-replication and haploidy of the chromosome in vivo. The data-carrying chromosome displayed highly pervasive transcriptional ability and transcribed hundreds of non-coding RNAs. In summary, this work explores the chromatin epigenetic state, chromatin structure and transcriptional landscape of an exogenous artificial chromosome. The results demonstrated that the exogenous artificial chromosome did form a chromatin structure and was not a naked and incompact plasmid, which strengthen our understanding of artificial chromosomes.
Project description:Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. However, all TIS methods described to date use short nucleotide sequence reads which cannot uniquely determine the locations of transposon insertions within repeating genomic sequences where the repeat units are longer than the sequence read length. To overcome this limitation, we have developed a TIS method using Oxford Nanopore sequencing technology that generates and uses long nucleotide sequence reads; we have called this method LoRTIS (Long Read Transposon Insertion-site Sequencing). This experiment data contains sequence files generated using Nanopore and Illumina platforms. Biotin1308.fastq.gz and Biotin2508.fastq.gz are fastq files generated from nanopore technology. Rep1-Tn.fastq.gz and Rep1-Tn.fastq.gz are fastq files generated using Illumina platform. In this study, we have compared the efficiency of two methods in identification of transposon insertion sites.
Project description:We used the nanopore Cas9 targeted sequencing (nCATS) strategy to specifically sequence 125 L1HS-containing loci in parallel and measure their DNA methylation levels using nanopore long-read sequencing. Each targeted locus is sequenced at high coverage (~45X) with unambiguously mapped reads spanning the entire L1 element, as well as its flanking sequences over several kilobases. The genome-wide profile of L1 methylation was also assessed by bs-ATLAS-seq in the same cell lines (E-MTAB-10895).
Project description:Anthrax is a zoonotic infection caused by the bacterium Bacillus anthracis (BA), a gram-positive, aerobic, spore-forming bacterium that can be misused as a biowarfare agent. The major patho-genicity factors of BA are encoded by genes located on two extrachromosomal plasmids, which are often targeted for specific identification of this pathogen. However, more recent findings show that these plasmids are not a unique feature of BA but can also occur in other Bacillus species. Furthermore, BA is a member of the Bacillus cereus group, a subgroup of closely related Bacilli. Due to the high genetic similarity within this group, it is a challenge to distinguish BA from other members of this group. In this study, we investigated if it is possible to identify species-specific and universally applicable marker peptides for BA. For this purpose, we applied a high-resolution mass spectrometry-based approach for 42 BA isolates. Together with the genomic sequencing data and by developing a bioinformatics data evaluation pipeline, which uses a database containing most of the publicly available protein sequences worldwide (UniParc), we were able to identify eleven universal marker peptides unique to BA, which are located on the chromosome and there-fore might overcome known problems like observable loss of plasmids in environmental species, plasmid loss during cultivation in the lab and that the virulence plasmids are not necessarily a unique feature of BA. The identified chromosomally encoded markers in this study could extend the small panel of already existing chromosomal targets and together with targets for the viru-lence plasmids may pave the way to an even more reliable identification of BA using genomics- as well as proteomics-based techniques.
Project description:Chromosomes and plasmids are two forms of genetic carriers. Exogenous yeast artificial chromosomes are also considered as yeast centromeric plasmids in many cases. Here, we used state-of-the-art sequencing technologies to comprehensively profile the genetic, epigenetic, transcriptional and proteomic characteristics of an exogenous data-carrying chromosome. We found that the data-carrying DNA formed active chromatin with high chromatin accessibility and H3K4 tri-methylation levels. We also confirmed that the data-carrying chromosome had a circular shape in the nucleus and was arranged in the Rabl configuration, which may contribute to the self-replication and haploidy of the chromosome in vivo. The data-carrying chromosome displayed highly pervasive transcriptional ability and transcribed hundreds of non-coding RNAs. In summary, this work explores the chromatin epigenetic state, chromatin structure and transcriptional landscape of an exogenous artificial chromosome. The results demonstrated that the exogenous artificial chromosome did form a chromatin structure and was not a naked and incompact plasmid, which strengthen our understanding of artificial chromosomes.
Project description:Nanopore Sequencing and assembly of Col-0 carrying seed coat expressed GFP and RFP transgenes flanking the centromere of chromosome 3 (CTL 3.9) - additionally, DNA methylation was derived using deepsignal-plant using these reads.