Targeted Enrichment of rRNA Gene Tandem Arrays for Ultra-Long Sequencing by Selective Restriction Endonuclease Digestion

Large regions of nearly identical repeats, such as the 45S ribosomal RNA (rRNA) genes of Nucleolus Organizer Regions (NORs), can account for major gaps in sequenced genomes. To assemble these regions, ultra-long sequencing reads that span multiple repeats have the potential to reveal sets of repeats that collectively have sufficient sequence variation to unambiguously define that interval and recognize overlapping reads. Because individual repetitive loci typically represent a small proportion of the genome, methods to enrich for the regions of interest are desirable. Here we describe a simple method that achieves greater than tenfold enrichment of Arabidopsis thaliana 45S rRNA gene sequences among ultra-long Oxford Nanopore Technology sequencing reads. This method employs agarose-embedded genomic DNA that is subjected to restriction endonucleases digestion using a cocktail of enzymes predicted to be non-cutters of rRNA genes. Most of the genome is digested into small fragments that diffuse out of the agar plugs, whereas rRNA gene arrays are retained. In principle, the approach can also be adapted for sequencing other repetitive loci for which gaps exist in a reference genome.

Rapid Detection of blaKPC-9 Allele from Clinical Isolates

The emergence of Klebsiella pneumoniae carbapenemase (KPC) nosocomial outbreaks related to specific blaKPC gene variants dictates the need for applicable diagnostic methods for allele discrimination. We report here a simple method of blaKPC-9 allele recognition based on a combination of endonuclease digestion analysis and PCR amplification using unique primers. K. pneumoniae isolates carrying the blaKPC gene were tested. Digestion with RsaI restriction endonuclease was found to efficiently differentiate the blaKPC-2 from the blaKPC-9 variants into two distinct groups of digestion patterns named KPC-2-like and KPC-9-like, respectively. An additional procedure, the amplification refractory mutation system (ARMS) method, was applied to identify the variant within the same group. The principles of this procedure could be developed to identify several blaKPC gene variants, as well as monitoring the spread and evolution of specific KPC variants within local geographical regions.

CaptureCompendium: a comprehensive toolkit for 3C analysis

ABSTRACTDNA folding within nuclei is a highly ordered process, with implications for gene regulation and development. An array of chromosome conformation capture (3C) methods have been developed to investigate how DNA is packaged within nuclei and to interrogate specific interactions. While these methods use different approaches to examine target loci (many-versus-all) or the entire genome (all-versus-all), they all rely on the core principle of endonuclease digestion and proximity-based ligation to re-arrange genomic order to reflect the three-dimensional nuclear conformation. This sequence reorganization creates novel chimeric DNA fragments which require specialist bioinformatic tools to analyze and visualize. Despite this need for specialist bioinformatic skills, the core biological importance of genome folding has seen widespread methodological uptake. To service the needs of experimentalists using the many-versus-all Capture-C family of methods we have developed CaptureCompendium; a toolkit of software to simplify the design, analysis and presentation of 3C experiments.

Higher-Order Organization Principles of Pre-translational mRNPs

SummaryCompared to noncoding RNAs (ncRNAs) such as rRNAs and ribozymes, for which high resolution structures abound, little is known about the tertiary structures of mRNAs. In eukaryotic cells, newly made mRNAs are packaged with proteins in highly compacted mRNPs, but the manner of this mRNA compaction is unknown. Here we developed and implemented RIPPLiT (RNA ImmunoPrecipitation and Proximity Ligation in Tandem), a transcriptome-wide method for probing the 3D conformations of RNAs stably-associated with defined proteins, in this case exon junction complex (EJC) core factors. EJCs multimerize with other mRNP components to form megadalton sized complexes that protect large swaths of newly synthesized mRNAs from endonuclease digestion. Unlike ncRNAs, mRNAs behave more like flexible polymers without strong locus-specific interactions. Polymer analysis of proximity ligation data for hundreds of mRNA species demonstrates that pre-translational mammalian mRNPs fold as linear rod-like structures with no strong propensity for 5’ and 3’ end interaction.

Schistosomiasis Induces Persistent DNA Methylation and Tuberculosis-specific Immune Changes

Epigenetic mechanisms, like DNA methylation, determine immune cell phenotype. To understand the epigenetic alterations induced by helminth co-infections, we evaluated the longitudinal effect of ascariasis and schistosomiasis infection on CD4+ T cell DNA methylation and the downstream tuberculosis (TB)-specific and BCG-induced immune phenotype. All experiments were performed on human primary immune cells from a longitudinal cohort of recently TB-exposed children. Compared to age-matched uninfected controls, children with active Schistosoma haematobium and Ascaris lumbricoides infection had 751 differentially DNA methylated genes with 72% hyper-methylated. Gene ontology pathway analysis identified inhibition of IFN-γ signaling, cellular proliferation, and the Th1 pathway. Targeted RT-PCR after methyl-specific endonuclease digestion confirmed DNA hyper-methylation of the transcription factors BATF3, ID2, STAT5A, IRF5, PPARg, RUNX2, IRF4 and NFATC1 and cytokines or cytokine receptors IFNGR1, TNFS11, RELT (TNF receptor), IL12RB2 and IL12B (p< 0.001; Sidak-Bonferroni). Functional blockage of the IFN-γ signaling pathway was confirmed with helminth-infected individuals having decreased up-regulation of IFN-γ-inducible genes (Mann-Whitney p < 0.05). Hypo-methylation of the IL-4 pathway and DNA hyper-methylation of the Th1 pathway was confirmed by antigen-specific multidimensional flow cytometry demonstrating decreased TB-specific IFN-γ and TNF and increased IL-4 production by CD4+ T cells (Wilcoxon signed rank P <0.05). In S.haematobium infected individuals, these DNA methylation and immune phenotypic changes persisted at least six months after successful deworming. This work demonstrates that helminth infection induces DNA methylation and immune perturbations that inhibit TB-specific immune control and that the duration of these changes are helminth-specific.

A Special Role for Neutrophil Extracellular Traps (NETs) and Neutrophils in the Prothombotic Nature of Heparin-Induced Thrombocytopenia

In response to infection and inflammation, neutrophils release NETs, histone-decorated nuclear DNA that ensnares bacteria, but also damages host tissues and promotes thrombosis. Platelet Factor 4 (PF4, CXCL4) is a CXC chemokine stored in high concentrations in platelet alpha-granules and released during platelet activation. Tetrameric PF4 has a very high affinity for heparans and other polyanionic molecules, including DNA. At the proper molar ratio, PF4 can form high molecular weight complexes with heparin and other heparans, and these complexes are antigenic targets for pathogenic HIT antibodies. In light of this information, we chose to investigate whether PF4 can interact with NETs and whether these complexes contribute to the prothrombotic nature of HIT. Using an ELISA assay, we confirm that PF4 binds to DNA to form HIT-like complexes just as it does with heparin following a similar bell-shaped curve of HIT antigenicity. Using immunofluorescence studies and confocal microscopy, we found that exogenous PF4 adheres readily to NET DNA. We then investigated PF4-NET interactions under intravascular flow conditions by using neutrophils isolated from healthy human donors to create NET-coated microfluidic channels through which we infused recombinant human PF4. We found that PF4 selectively adhered to extracellular NET fibers but did not bind to the surface of intact neutrophils. We next noted that PF4 infusion led to a change in NET morphology with compaction of the extracellular DNA to approximately 30% of the original area (p<0.001, N=10 per arm). KKO, a monoclonal anti-PF4-heparin antibody, bound readily to NETs following PF4 incubation, indicating that PF4-NET complexes are antigenic. Of note, KKO binding did not induce additional NET compaction. We then observed that while NETs were highly susceptible to endonuclease digestion prior to PF4 incubation, PF4-NET complexes developed resistance to endonuclease digestion. PF4-NET complex incubation with either KKO or HIT IgG isolated from patient samples further enhanced resistance to endonuclease digestion by >2-fold (p<0.001, N=2 studies, 5 NETS per study), while incubation with a polyclonal anti-PF4 antibody did not have this effect. We also show that neutrophils bind readily to endothelial cells when whole blood was flowed through microfluidic channels lined with TNFα-injured endothelium. The number of adherent neutrophils increased markedly when KKO was added to the whole blood, but no significant changes were observed when an isotype control antibody was included. These findings suggest that neutrophils may contribute to thrombosis in HIT through three sequential steps: (1) HIT-antibody activated neutrophils selectively bind to injured endothelium, increasing the numbers of localized neutrophils at sites of thrombus. (2) Subsequently released NETs are bound by PF4 and HIT antibodies, generating immunogenic complexes that are likely prothrombotic, and finally, (3) PF4 and HIT antibody binding induces resistance to endonuclease digestion leading to a prolongation of NET half-life and an increased opportunity to contribute to clot formation. We believe these data support a set of mechanisms by which neutrophils can contribute to the observed prothrombotic nature of HIT, including a novel NET stabilization process. Further in vivo mouse models will now be pursued to confirm these findings. Disclosures No relevant conflicts of interest to declare.

Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides

Biotinylation of deoxyguanosine at an abasic site in double-stranded oligodeoxynucleotides was studied. The biotinylation of deoxyguanosine is achieved by copper-catalyzed click reaction after the conjugation of the oligodeoxynucleotide with 2-oxohex-5-ynal. The biotinylation enables visualization of the biotinylated oligodeoxynucleotides by chemiluminescence on a nylon membrane. In order to investigate the biotinylated site, the biotinylated oligodeoxynucleotides were amplified by the DNA polymerase chain reaction. Replacement of guanine opposing the abasic site with adenine generated by the activity of the terminal deoxynucleotidyl transferase of DNA polymerase was detected by DNA sequencing analysis and restriction endonuclease digestion. This study suggests that 2-oxohex-5-ynal may be useful for the detection of the unpaired deoxyguanosine endogenously generated at abasic sites in genomic DNA.