High GC Content Research Articles

Development of specific monoclonal antibodies for the detection of natural chicken tumor necrosis factor-alpha

Tumor necrosis factor alpha (TNF-α) is an important proinflammatory cytokine and the only known cytokine that can directly kill tumor cells. Unlike mammalian counterparts, chicken TNF-α (chTNF-α) gene has not been identified until very recently due to its high GC content (∼70%) and long GC fragments. The biological functions of this newly-identified cytokine and its detection methods remain to be further investigated. In this study, the extracellular domain of chTNF-α was cloned into prokaryotic vector after codon optimization and recombinant chTNF-α protein was expressed. Subsequently, using recombinant chTNF-ɑ as immunogen, rabbit polyclonal antibody (pAb) and eight clones of mouse anti-chTNF-ɑ monoclonal antibodies (mAbs) were produced, respectively. Both the pAb and mAbs specifically recognized recombinant chTNF-ɑ expressed in E.coli and transfected COS-7 cells. Further mapping the antigenic region showed that all the mAbs recognized a region of amino acid residues 195–285 of chTNF-ɑ. Furthermore, an antigen-capture enzyme-linked immunosorbent assay for the detection of chTNF-ɑ was established using one mAb and the pAb. This assay showed no cross-reactivity with irrelevant Trx-fused antigens and could detect natural chTNF-ɑ expressed by mitogen-activated chicken splenocytes in a dose-dependent manner, with a detection limit of 1 ng/mL. Collectively, our results indicated that the mAbs and pAb against chTNF-α are specific and could be used for the study of the biological functions of chTNF-ɑ and the detection of chTNF-ɑ.

Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin.

Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) invitro and onto rDNA invivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at the melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment. In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100-500bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.

Intracellular common gardens reveal niche differentiation in transposable element community during bacterial adaptive evolution

The distribution and abundance of transposable elements across the tree of life have significantly shaped the evolution of cellular organisms, but the underlying mechanisms shaping these ecological patterns remain elusive. Here we establish a “common garden” approach to study causal ecological interactions between a xenogeneic conditional lethal sacB gene and the community of transposable insertion sequences (ISs) in a multipartite prokaryote genome. Xenogeneic sacB of low, medium, or high GC content was individually inserted into three replicons of a model bacterium Sinorhizobium fredii, and exhibited replicon- and GC-dependent variation in genetic stability. This variation was largely attributable to multidimensional niche differentiation for IS community members. The transposition efficiency of major active ISs depended on the nucleoid-associated xenogeneic silencer MucR. Experimentally eliminating insertion activity of specific ISs by deleting MucR strongly demonstrated a dominant role of niche differentiation among ISs. This intracellular common garden approach in the experimental evolution context allows not only for evaluating genetic stability of natural and synthetic xenogeneic genes of different sequence signatures in host cells but also for tracking and testing causal relationships in unifying ecological principles in genome ecology.

Sequence-based detection and typing procedures for Burkholderia mallei: Assessment and prospects.

Although glanders has been eradicated in most of the developed world, the disease still persists in various countries such as Brazil, India, Pakistan, Bangladesh, Nepal, Iran, Bahrain, UAE and Turkey. It is one of the notifiable diseases listed by the World Organization for Animal Health. Occurrence of glanders imposes restriction on equestrian events and restricts equine movement, thus causing economic losses to equine industry. The genetic diversity and global distribution of the causing agent, Burkholderia (B.) mallei, have not been assessed in detail and are complicated by the high clonality of this organism. Among the identification and typing methods, PCR-based methods for distinguishing B. mallei from its close relative B. pseudomallei as well as genotyping using tandem repeat regions (MLVA) are established. The advent and continuous advancement of the sequencing techniques and the reconstruction of closed genomes enable the development of genome guided epidemiological tools. For achieving a higher genomic resolution, genotyping methods based on whole genome sequencing data can be employed, like genome-wide single nucleotide polymorphisms. One of the limitations in obtaining complete genomic sequences for further molecular characterization of B. mallei is its high GC content. In this review, we aim to provide an overview of the widely used detection and typing methods for B. mallei and illustrate gaps that still require development. The genomic features of Burkholderia, their high homology and clonality will be first described from a comparative genomics perspective. Then, the commonly used molecular detection (PCR systems) and typing systems (e.g., multilocus sequence typing, variable number of tandem repeat analysis) will be presented and put in perspective with recently developed genomic methods. Also, the increasing availability of B. mallei genomic sequences and evolution of the sequencing methods offers exciting prospects for further refinement of B. mallei typing, that could overcome the difficulties presently encountered with this particular bacterium.

Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Its Subsequent Fermentation for Optimized Production.

The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the Gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40% of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60%, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC Gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 25years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article, an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans as a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Stable and efficient marker-less integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR23A-based system will be explained. Finally, a basic protocol for bench-top bioreactor experiments which can form the start in the production process optimization and up-scaling will be provided.

Economizing marker assisted selection through cost-effective assay of sd1 gene in rice (Oryza sativa)

PCR amplification and detection of GC rich sequences in DNA is a challenge due to formation of secondary structures which resist denaturation, thereby stalling Taq DNA polymerases as well as affecting primer annealing. Presently, high fidelity Taq DNA polymerase is used for amplifying long and GC-rich fragments, while dimethyl sulfoxide (DMSO) has also been suggested as an additive in Polymerase Chain Reaction (PCR) mix to avoid formation of secondary structures in templates containing high GC content. In the present study, the amplification efficiency of normal Taq DNA polymerase with 5% DMSO as compared to high fidelity Taq DNA polymerase has been applied for better amplification of fragments with high GC content of sd1 gene in rice. Normal Taq DNA polymerase with 5% DMSO displayed better and reproducible results as compared to platinum DNA polymerase for the amplification of gene sd1, thereby successfully detecting the dominant wild type allele governing tall plant height from the recessive semi-dwarf allele. There were no adverse effects of 5% DMSO in PCR amplification and the amplified fragments improved significantly indicating the improvement in amplification efficiency. The modified PCR protocol including DMSO provides a cost effective and cheaper alternative for reliable assessment of the genomic differences in GCrich regions of the gene. This will help in eliminating the need for costly high fidelity Taq DNA polymerase, thereby helping in economizing marker assisted selection.

Core Proteomics and Immunoinformatic Approaches to Design a Multiepitope Reverse Vaccine Candidate against Chagas Disease.

Chagas disease is a tropical ailment indigenous to South America and caused by the protozoan parasite Trypanosoma cruzi, which has serious health consequences globally. Insect vectors transmit the parasite and, due to the lack of vaccine availability and limited treatment options, we implemented an integrated core proteomics analysis to design a reverse vaccine candidate based on immune epitopes for disease control. Firstly, T. cruzi core proteomics was used to identify immunodominant epitopes. Therefore, we designed the vaccine sequence to be non-allergic, antigenic, immunogenic, and to have better solubility. After predicting the tertiary structure, docking and molecular dynamics simulation (MDS) were performed with TLR4, MHC-I, and MHC-II receptors to discover the binding affinities. The final vaccine design demonstrated significant hydrogen bond interactions upon docking with TLR4, MHC-I, and MHC-II receptors. This indicated the efficacy of the vaccine candidate. A server-based immune simulation approach was generated to predict the efficacy. Significant structural compactness and binding stability were found based on MDS. Finally, by optimizing codons on Escherichia coli K12, a high GC content and CAI value were obtained, which were then incorporated into the cloning vector pET2+ (a). Thus, the developed vaccine sequence may be a viable therapy option for Chagas disease.

IKZF1 DELETIONS IN B-ALL: FROM GENETIC BASIS TO DIAGNOSTIC ENHANCEMENT

Because IKZF1 deletions are associated with an increased risk of relapse in patients with B-cell precursor acute lymphoblastic leukemia (B-ALL), the development of methods for rapid and accurate detection of this alteration has a great clinical impact. Although multiplex ligation-dependent probe amplification (MLPA) has been widely used for the evaluation of IKZF1 deletions, multiplex (M)-PCR is a valuable tool for the validation of those IKZF1 deletions. However, this method is still limited to detect recurrent intragenic deletions (Δ2-3, Δ2-7, Δ2-8, Δ4-7, Δ4-8). Therefore, here we evaluate the breakpoint map of IKZF1 deletions with a special focus on non-recurrent deletions to update methodologies for the diagnosis of this genetic alteration. Moreover, we explore the genetic features underlying those IKZF1 deletions to infer its molecular pathogenesis. This study included four international cohorts of pediatric and adult patients with B-ALL. IKZF1 deletions were identified by MLPA (P335 and P202 assays) and M-PCR. Non-recurrent deletions were verified by next-generation sequencing using the Illumina DNA Prep with Enrichment assay. We also revised previously published data regarding IKZF1 deletions in B-ALL for compiling breakpoint sequence information of these deletions. Sequence data from our cohorts and literature were used in further analyses. After identifying the breakpoint clusters, we performed an agnostic motif search using MEME and annotated the presence of filler DNA or microhomologies at the junctions of IKZF1 deletions. A total of 1,472 B-ALL samples were screened, and IKZF1 deletions were found in 16% of these patients. Although non-recurrent deletions were associated with 1% of the total cohort, it represents 8% of all IKZF1 deletions. The analysis of breakpoint sequences at DNA level revealed that non-recurrent IKZF1 deletions comprises miscellaneous alterations, but the majority of them encompases Δ1 and Δ1-3. Considering that IKZF1 exon 1 deletions are more difficult to reliably detect by MLPA due to the high GC content (>50%) of this region, we mapped several exon 1 deletions at DNA level and identified one commonly deleted region which is ideal for designing MLPA probes. Next, we searched for motifs, additional nucleotides/microhomologies associated with IKZF1 deletion sequences. We verified an enrichment of the 5’-CACACAGTG-3’sequence within 21 out of 24 breakpoint clusters of IKZF1 deletion (E-value: 2.9e-008). Additionally, the filler DNA within the junctions of IKZF1 deletions presented a higher GC nucleotide content at these clusters as compared to the deletions outside of them (71% vs. 56%). This study provides the genetic landscape of IKZF1 deletions in B-ALL. Although rare types of deletion may be identified at the routine diagnosis, they should be considered after using adapted MLPA protocols, including additional probes within the common deleted region nearby IKZF1 exon 1. We also verified the occurrence of the heptamer sequence and an enrichment of GC nucleotides exclusively within breakpoint clusters, suggesting that RAG recombination and TdT activity may promote most IKZF1 deletions, although rare types of alterations may be associated with another molecular mechanism of leukemogenesis.

ANALYTICAL PERFORMANCE OF A DNA/RNA-BASED NEXT-GENERATION SEQUENCING (NGS) PANEL FOR MYELOID NEOPLASMS

Myeloid neoplasms (MNs) are a heterogeneous group of neoplasms including acute myeloid leukemia, myelodysplastic syndrome, myeloproliferative neoplasms and myelodysplastic syndrome/myeloproliferative neoplasms. MNs show a complex genetic basis, and the number of alterations to be analyzed for risk diagnostic, stratification and management of patients increases continuously. Consequently, there is a demand for multiple molecular and cytogenetic techniques with the aim of detecting a wide variety of genetic abnormalities. Since conventional techniques are insufficient to study all such abnormalities, the NGS panel has become a useful tool for broad characterization of the spectrum of mutations in MNs. However, before NGS panels can be introduced in the laboratory routine, their performance should be evaluated. In this context, we describe the analytical performance of the Oncomine™Myeloid Research Assay (OMA) (Thermo Fisher), a DNA/RNA-based amplicon sequencing panel, that evaluated simultaneously 40 genes (17 full genes and 23 hotspots genes) and 29 fusions drivers genes (over 600 partners) known to be recurrently mutated in MNs. Commercial controls Seraseq Myeloid Mutation DNA and the Seraseq Myeloid Fusion RNA were used for sensitivity, specificity, accuracy, repeatability, reproducibility and limit of detection (LoD) testing. Prospective sequencing was performed for samples from 34 patients presenting with suspected or confirmed MN. Libraries was prepared by manual method and sequenced by the Ion S5 Sequencer. Data obtained were analysed on Ion Reporter 5.18 software. The OMA assay showed good performance in terms of depth of coverage, on-target reads, and uniformity. The mean reads lenght ranged from 223 to 231 bp for DNA sample and from 88 to 124 bp for RNA, within the expected standard for the panel. The panel achieved 98,8% and 100% concordance with DNA and RNA controls, reaching analytical sensitivity of 99% and 100% for DNA and RNA, respectively. Regarding the type of DNA mutation: single nucleotide variants (SNP), indels or indels hot spots, the sensitivity found was 100%, 96% and 95%, respectively. The accuracy and analytical specificity was 100% for DNA panel. The LoD was 5% variant allele fraction (VAF) for DNA (SNP and indel) and 1-log reduction from initial number of reads counts for RNA fusion genes. Repeatability and reproducibility were 98% for DNA and 100% for RNA. However, the coefficient of variation (CV) within-run and between-run in VAF was >20% for large indels in CALR, FLT3 (ITD) and SRSF2 . The vertical coverage of each sample was inspected with a coverage threshold of 100X. Some amplicons in the assay failed repeatedly (ASXL1_1.875, BCOR_8.202477, BRAF_18.30633, CEBPA_1.1.86676, PRPF8_12.173121, SH2B3_1.85663). However, the OMA is unable to detect specific variants in ASXL1 (c.1900_1922del23 and c.1934_1935insG) and CEBPA (c.68_69insC). This is a known limitation of the Ion Torrent chemistry for accurate sequencing of large homopolymers and high GC content regions. Of the 34 patient samples, 28 (87.5%) had at least one reportable variant. A total of 58 DNA variants and six gene fusions were detected. Some variants were confirmed by orthogonal techniques. In conclusion, OMA is a highly accurate and reproducible NGS panel for the detection of common genetic abnormalities in MNs.

Delineating species boundary through DNA barcodes ofsome fishes inhabiting river Murti in the foothills of Darjeeling Himalayas

The aim of the present study was to establish a global bioidentification system in the form of a reference sequencelibrary where fish species inhabiting river Murti were delineated by a particular sequence denoted as genetic‘barcode’.40 species representing 27 genera, 16 families and 5 orders were collected along different altitudinal gradients if river Murti. Mitochondrial DNA was isolated from the samples followed by amplification of COI gene, alignment by CLUSTAL W and submission to GenBank. The average nucleotide frequencies were calculated whereall the fishes showed relatively higher GC content at second codon position. Haplotypes showed gradually decreasinggenetic distance within orders, families, genera with very low genetic distance within species indicating markedgenetic divergence beyond species boundaries. The average transitional pairs were found more frequent than averagetransversional pairs. The Neighbour Joining tree revealed distinct clusters formed by members of order Cypriniformes, Siluriformes, Perciformes, Synbranchiformes and Beloniformes, where the conspecific individualswere always found to cluster under the same node supported by high bootstrap value, while dissimilar species wereclustered under separate nodes, ensuring unambiguous identification of species. The research work suggests that COI barcoding can be taken up as a sensible approach to eliminate obscurity in the identification of the fish fauna withapplications in its management and conservation of the species inhabiting in specific microhabitats. Keywords: COI gene, DNA Barcode, sequence library, unambiguous identification, conservation.

Bioprospecting of actinomycetes for anti-bacterial potential: A Review

Actinomycetes are gram-positive bacteria that have a high GC content compared to their counterparts. Actinomycetes were capable of producing a broad range of secondary metabolites and many of these were anti-bacterial agents. Antibiotics have been known to play a vital role in therapeutics and the clinical management of various diseases. Actinomycetes have contributed more than 70% of known antibiotics in the market today. Apart from antibiotics, actinomycetes also produce several enzymes, enzyme inhibitors, anticancer and anti-diabetic secondary metabolites. Currently, actinomycetes are being studied for medicinal, pharmaceutical, industrial, environmental and bioremediation applications. The current study focuses on a variety of antibacterial agents derived from actinomycetes and new antibiotics under clinical trial along with their mode of action. It also describes the genome mining methods for screening actinomycetes isolates for biosynthetic gene clusters and specific antibacterial secondary metabolites.

Genomic Evolution of ST11 Carbapenem-Resistant Klebsiella pneumoniae from 2011 to 2020 Based on Data from the Pathosystems Resource Integration Center.

(1) Objective: ST11 carbapenem-resistant Klebsiella pneumoniae (CRKP) is widespread throughout the world, and the mechanisms for the transmission and evolution of major serotypes, ST11-KL47 and ST11-KL64, were analyzed to investigate the global distribution and evolutionary characteristics of ST11 CRKP; (2) Methods: The Pathosystems Resource Integration Center (PATRIC) database was downloaded and all K. pneumoniae from 2011 to 2020 were screened to obtain ST11 CRKP genome assemblies with basic information. The relationship of serotype evolution between KL47 and KL64 was then investigated using statistical and bioinformatic analysis; (3) Results: In total, 386 ST11 CRKP isolates were included for analysis. Blood (31.09%, 120/386), respiratory tract (23.06%, 89/386), and feces (20.21%, 78/386) were the major sources of samples. China was the leading country where ST11 CRKP was isolated. KL47 and KL64 were found to be the most prevalent serotypes. ST11-KL64 CRKP [median 78(P25~P75: 72~79.25)] had remarkably more virulence genes than the KL47 [median 63(P25~P75: 63~69)], and the distinction was statistically significant (p < 0.001). A differential comparison of virulence genes between KL47 and KL64 revealed 35 differential virulence genes, including rmpA/rmpA2, iucABCD, iutA, etc. The comparison of the recombination of serotype-determining regions between the two serotypes revealed that KL64 CRKP carried more nucleotide sequences in the CD1-VR2-CD2 region than KL47 CRKP. More nucleotide sequences added approximately 303 base pairs (bp) with higher GC content (58.14%), which might facilitate the evolution of the serotype toward KL64; (4) Conclusions: KL47 and KL64 have become the predominant serotypes of ST11 CRKP. KL64 CRKP carries more virulence genes than KL47 and has increased by approximately 303 bp through recombinant mutations, thus facilitating the evolution of KL47 to KL64. Stricter infection prevention and control measures should be developed to deal with the epidemic transmission of ST11-KL64 CRKP.

P14.04.A TrackingTERTpromoter andIDH1mutations in liquid biopsies - suitable biomarkers for disease monitoring in glioma patients?

Abstract Background Mutations within the telomerase reverse transcriptase promoter (TERTprom) and isocitrate dehydrogenase (IDH) account for the most common genetic alterations in gliomas. Each of these mutations impact clinicopathologic diagnosis and course of diseases. While TERTprom mutations are frequently detected in glioblastoma, IHD mutations are assigned to astrocytoma of grade 2-4, thus mostly associated with better prognosis. In the era of precision oncology, molecular profiling and continuous monitoring of treatment response or relapse are of increasing importance. Accordingly, this study aims to detect TERTprom and IDH mutations in plasma-derived cell-free (cf)DNA of gliomas. The mutant allele frequencies (MAF) will be compared retrospectively to clinico-pathological parameters including extent of resection and tumor progression. Material and Methods Digital droplet PCR (ddPCR) analyses were performed using the QX200TM Digital Droplet System from BioRad. First, to evaluate probes for ddPCR, genomic DNA of several brain tumor cell models (n=6) and tumor tissue (n=1), as well as cfDNA of plasma (n=3) from samples with known TERTprom and IDH mutation status was investigated. For detection of IDH mutations, the unique assay ID dHSaMDV2010055 (IDH1p.R132H) and for TERTprom mutations the TaqMan dPCR Liquid Biopsy Assays for C228T (Hs000000092) and C250T (Hs000000093) were used. The results of ddPCR were analyzed with QuantaSoftTM software and the MAF was calculated Results To validate the detection method for IDH1R132H, we analyzed the MAF in one tissue and corresponding plasma sample of a confirmed IDH1-mutated astrocytoma. In addition, plasma from one astrocytoma grade 2-3 as well as from an IDH1-mutated glioblastoma was tested. Interestingly, both astrocytoma cases exhibited undetectable or very low MAF ranging from 0.1 to 1% in tissue as well as in plasma samples, while in plasma from the high-grade glioblastoma case, IDH1R132H was detected with a frequency of 1.9%. Due to the high GC content of the TERT promoter region, amplification steps are challenging. Accordingly, we first optimized ddPCR conditions for C228T and C250T probes by adding 7-deaza-2-deoxyguanosine-5-triphosphate (7-ddGTP) in varying concentrations to each ddPCR reaction. When using 4µM of 7-ddGTP per sample, a clear separation between mutant and wild-type droplets was reached, detecting MAF between 36-63% in DNA from cell culture models. Conclusion Within this pilot study we optimized the ddPCR method for the detection of IDH1R132H and TERTprom mutations in plasma and tissue samples. Subsequently, we hypothesize that these mutations are suitable liquid biomarkers correlating with extent of resection and tumor progression in gliomas.

Capturing Structural Variants of Herpes Simplex Virus Genome in Full Length by Oxford Nanopore Sequencing.

ABSTRACTGenome sequencing and assembly of viral genomes within the Herpesviridae family, particularly herpes simplex virus (HSV), have been challenging due to the large size (~154 Kb), high GC content (68%), and nucleotide variations arising during replication. Oxford Nanopore Technology (ONT) has been successful in obtaining read lengths ranging from 100 Kb up to 2.3 Mb. We have optimized DNA extraction and sequencing with ONT to capture the whole genome of HSV-1 as a single read. Although previous studies described the presence of four different genome isomers of HSV, we provided the first report on capturing all four variants’ full-length genome as single reads. These isomers were found to be present in almost equal proportion in the sequenced DNA preparation.IMPORTANCE With the advent of next-generation sequencing platforms, genome sequencing of viruses can be performed in a relatively shorter time frame in even the most austere conditions. Ultralong read sequencing platforms, such as Oxford Nanopore Technology (ONT), have made it possible to capture the full-length genome of DNA viruses as a single read. By optimizing ONT for this purpose, we captured the genome (~154 Kb) of a clinical strain of herpes simplex virus 1 (HSV-1). Additionally, we captured full-length sequences of the four isomers of lab-grown HSV-1 virus and were able to determine the frequency of each within the isogenic population. This method will open new directions in studying the significance of these isomers and their clinical relevance to HSV-1 infections. It will also improve basic studies on the recombination and replication of this virus.

RNA helicase-dependent gene looping impacts messenger RNA processing.

DDX5 and DDX17 are DEAD-box RNA helicase paralogs which regulate several aspects of gene expression, especially transcription and splicing, through incompletely understood mechanisms. A transcriptome analysis of DDX5/DDX17-depleted human cells confirmed the large impact of these RNA helicases on splicing and revealed a widespread deregulation of 3′ end processing. In silico analyses and experiments in cultured cells showed the binding and functional contribution of the genome organizing factor CTCF to chromatin sites at or near a subset of DDX5/DDX17-dependent exons that are characterized by a high GC content and a high density of RNA Polymerase II. We propose the existence of an RNA helicase-dependent relationship between CTCF and the dynamics of transcription across DNA and/or RNA structured regions, that contributes to the processing of internal and terminal exons. Moreover, local DDX5/DDX17-dependent chromatin loops spatially connect RNA helicase-regulated exons with their cognate promoter, and we provide the first direct evidence that de novo gene looping modifies alternative splicing and polyadenylation. Overall our findings uncover the impact of DDX5/DDX17-dependent chromatin folding on pre-messenger RNA processing.

Reorganization of the Landscape of Translated mRNAs in NSUN2-Deficient Cells and Specific Features of NSUN2 Target mRNAs.

The RNA cytosine C5 methyltransferase NSUN2 has a variety of RNA substrates and plays an important role in mRNA metabolism. NSUN2 binds to specific sequences enriched in exosomal mRNAs, suggesting its possible involvement in the sorting of mRNAs into exosomes. We applied the photoactivatable.4-thiouridine-enhanced cross-linking and immunoprecipitation assay involving high-throughput RNA sequencing (RNA-seq) to HEK293T cells to determine NSUN2 mRNA targets. NSUN2 cross-linking sites were found in more than one hundred relatively abundant mRNAs with a high GC content and a pronounced secondary structure. Then, utilizing RNA-seq for the total and polysome-associated mRNA from HEK293T cells with and without the knockdown of NSUN2, we identified differentially expressed genes, as well as genes with altered translational efficiency (GATEs). It turned out that the up-regulated GATE mRNAs were much shorter on average than the down-regulated ones, and their GC content was higher; moreover, they contained motifs with C residues located in GC-rich environments. Our findings reveal the specific features of mRNAs that make them potential targets for NSUN2 and expand our understanding of the role of NSUN2 in controlling translation and, possibly, in mRNA sorting into exosomes implemented through the methylation of cytosine residues.

Oligo replication advantage driven by GC content and Gibbs free energy.

Large scale DNA oligo pools are emerging as a novel material in a variety of advanced applications. However, GC content and length cause significant bias in amplification of oligos. We systematically explored the amplification of one oligo pool comprising of over ten thousand distinct strands with moderate GC content in the range of 35-65%. Uniqual amplification of oligos result to the increased Gini index of the oligo distribution while a few oligos greatly increased their proportion after 60 cycles of PCR. However, the significantly enriched oligos all have relatively high GC content. Further thermodynamic analysis demonstrated that a high value of both GC content and Gibbs free energy could improve the replication of specific oligos during biased amplification. Therefore, this double-G (GC content and Gibbs free energy) driven replication advantage can be used as a guiding principle for the sequence design for a variety of applications, particularly for data storage.

Decoding regulatory associations of G-quadruplex with epigenetic and transcriptomic functional components.

G-quadruplex (G4) has been previously observed to be associated with gene expression. In this study, we performed integrative analysis on G4 multi-omics data from in-silicon prediction and ChIP-seq in human genome. Potential G4 sites were classified into three distinguished groups, such as one group of high-confidence G4-forming locations (G4-II) and groups only containing either ChIP-seq detected G4s (G4-I) or predicted G4 motif candidates (G4-III). We explored the associations of different-confidence G4 groups with other epigenetic regulatory elements, including CpG islands, chromatin status, enhancers, super-enhancers, G4 locations compared to the genes, and DNA methylation. Our elastic net regression model revealed that G4 structures could correlate with gene expression in two opposite ways depending on their locations to the genes as well as G4-forming DNA strand. Some transcription factors were identified to be over-represented with G4 emergence. The motif analysis discovered distinct consensus sequences enriched in the G4 feet, the flanking regions of two groups of G4s. We found high GC content in the feet of high-confidence G4s (G4-II) when compared to high TA content in solely predicted G4 feet of G4-III. Overall, we uncovered the comprehensive associations of G4 formations or predictions with other epigenetic and transcriptional elements which potentially coordinate gene transcription.

Highly Sensitive EGFRvIII Detection in Circulating Extracellular Vesicle RNA of Glioma Patients.

Liquid biopsy offers an attractive platform for noninvasive tumor diagnosis, prognostication, and prediction of glioblastoma clinical outcomes. Prior studies report that 30% to 50% of GBM lesions characterized by EGFR amplification also harbor the EGFRvIII mutation. A novel digital droplet PCR (ddPCR) assay for high GC content amplicons was developed and optimized for sensitive detection of EGFRvIII in tumor tissue and circulating extracellular vesicle RNA (EV RNA) isolated from the plasma of patients with glioma. Our optimized qPCR assay detected EGFRvIII mRNA in 81% [95% confidence interval (CI), 68%-94%] of EGFR-amplified glioma tumor tissue, indicating a higher than previously reported prevalence of EGFRvIII in glioma. Using the optimized ddPCR assay in discovery and blinded validation cohorts, we detected EGFRvIII mutation in 73% (95% CI, 64%-82%) of patients with a specificity of 98% (95% CI, 87%-100%), compared with qPCR tumor tissue analysis. In addition, upon longitudinal monitoring in 4 patients, we report detection of EGFRvIII in the plasma of patients with different clinical outcomes, rising with tumor progression, and decreasing in response to treatment. This study demonstrates the feasibility of detecting EGFRvIII mutation in plasma using a highly sensitive and specific ddPCR assay. We also show a higher than previously reported EGFRvIII prevalence in glioma tumor tissue. Several features of the assay are favorable for clinical implementation for detection and monitoring of EGFRvIII-positive tumors.

Whole human genome 5’-mC methylation analysis using long read nanopore sequencing

ABSTRACT Methylation microarray and bisulphite sequencing are often used to study 5’-methylcytosine (5’-mC) modification of CpG dinucleotides in the human genome. Although both technologies produce trustworthy results, the evaluation of the methylation status of CpG sites suffers from the potential side effects of DNA modification by bisulphite and/or the ambiguity of mapping short reads in repetitive and highly homologous genomic regions, respectively. Nanopore sequencing is an attractive alternative for the study of 5’-mC since it allows sequencing of native DNA molecules, whereas the long reads produced by this technology help to increase the resolution of those genomic regions. In this work, we show that nanopore sequencing with 10X coverage depth, using DNA from a human cell line, produces 5’-mC methylation frequencies consistent with those obtained by 450k microarray, digital restriction enzyme analysis of methylation, and reduced representation bisulphite sequencing. High correlation between methylation frequencies obtained by nanopore sequencing and the other methodologies was also noticeable in either low or high GC content regions, including CpG islands and transcription start sites. We also showed that a minimum of five reads per CpG yields strong correlations (>0.89) in replicate nanopore sequencing runs and an almost uniform linearity of the methylation frequency variation between zero and one. Furthermore, nanopore sequencing was able to correctly display methylation frequency patterns based on genomic annotations of CpG regions. These results demonstrate that nanopore sequencing is a fast, robust, and reliable approach to the study of 5’-mC in the human genome with low coverage depth.