scholarly journals Comparison of DNA and RNA sequencing of total nucleic acids from human cervix for metagenomics

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Laila Sara Arroyo Mühr ◽  
Joakim Dillner ◽  
Agustin Enrique Ure ◽  
Karin Sundström ◽  
Emilie Hultin

AbstractAlthough metagenomics and metatranscriptomics are commonly used to identify bacteria and viruses in human samples, few studies directly compare these strategies. We wished to compare DNA and RNA sequencing of bacterial and viral metagenomes and metatranscriptomes in the human cervix. Total nucleic acids from six human cervical samples were subjected to DNA and RNA sequencing. The effect of DNase-treatment before reverse transcription to cDNA were also analyzed. Similarities and differences in the metagenomic findings with the three different sequencing approaches were evaluated. A higher proportion of human sequences were detected by DNA sequencing (93%) compared to RNA sequencing without (76%) and with prior DNase-treatment (11%). On the contrary, bacterial sequences increased 17 and 91 times. However, the number of detected bacterial genera were less by RNA sequencing, suggesting that only a few contribute to most of the bacterial transcripts. The viral sequences were less by RNA sequencing, still twice as many virus genera were detected, including some RNA viruses that were missed by DNA sequencing. Metatranscriptomics of total cDNA provided improved detection of mainly transcribed bacteria and viruses in cervical swabs as well as detection of RNA viruses, compared to metagenomics.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 153-153
Author(s):  
Denise Lau ◽  
Alan Chang ◽  
Jason Perera ◽  
Ariane Lozac'hmeur ◽  
Alexandria Bobe ◽  
...  

153 Background: In the past decade, immunotherapy has emerged as an important new modality in cancer treatment. However, studies have shown that only a fraction of patients will experience any clinical benefit when treated with immune checkpoint blockade drugs. Given the cost and potent adverse events associated with immunotherapy, the need for effective biomarkers is clear. We sought to understand the role of key immunotherapy biomarkers, like tumor mutational burden (TMB), microsatellite instability (MSI), and PD-L1 immunohistochemistry (IHC), in the context of the greater immunogenomic landscape of solid tumors in patients. Methods: We analyzed data from a cohort of 500 patients across 10 cancer types who received the Tempus xT 595 gene targeted DNA sequencing assay and whole transcriptome sequencing assay as part of their clinical care. We determined the TMB, MSI status, and neoantigen load for each sample using the DNA sequencing data. We used the RNA expression data to evaluate immune activation and tumor infiltration by determining the expression of inflammatory gene signatures and estimating the relative proportion of key immune cell types. Results: Integrative analysis of the DNA and RNA sequencing data showed that the immunogenicity of the tumor, as measured by TMB or neoantigen load, correlates with levels of immune activation and tumor infiltration. Inflammatory immune cells, like CD8 T cells and M1 polarized macrophages, were significantly higher in TMB-high samples; while non-inflammatory immune cells, like monocytes, were significantly lower in TMB-high samples. Additionally, samples could be clustered into immunologically active “hot” tumors or immunologically silent “cold” tumors based on gene expression. The immunologically “hot” population was enriched for samples that were TMB-high, MSI-high or PD-L1 IHC positive. Conclusions: Paired next generation DNA and RNA sequencing assays allows for the identification of patients that have immunologically active tumors that lack traditional immunotherapy biomarkers. These patients represent an interesting new population who may potentially benefit from immunotherapy.


1964 ◽  
Vol 119 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Heather Donald Mayor

The nucleic acids produced intracellularly during the replication cycles of both DNA and RNA viruses can now be identified rapidly using a sensitized procedure based on staining with the fluorochrome acridine orange. Cellular DNA, viral DNA (both single and double stranded forms), cellular RNA, and RNA arising as a result of viral stimulus can be differentiated. The intracellular development of virus specific DNA, RNA, and protein has been studied in monkey kidney cells infected with adenoviruses types 3 and 7. It has been possible to detect a labile RNA in the nucleus from 16 to 20 hours after inoculation. When the cultures are treated with puromycin at this time, this RNA can be accumulated under certain conditions in the nucleus and demonstrated cytochemically. At the same time the production of specific viral protein as determined by staining with fluorescein-labeled antibodies is markedly inhibited. However, intranuclear double stranded DNA continues to be formed for a time. When puromycin is added to the system early in the eclipse period virus-specific DNA and labile RNA cannot be detected.


2018 ◽  
Author(s):  
Miranda E. Pitt ◽  
Son H. Nguyen ◽  
Tânia P.S. Duarte ◽  
Mark A.T. Blaskovich ◽  
Matthew A. Cooper ◽  
...  

ABSTRACTKlebsiella pneumoniae frequently harbour multidrug resistance and current methodologies are struggling to rapidly discern feasible antibiotics to treat these infections. While rapid DNA sequencing has been proposed for prediction of resistance profile; the role of rapid RNA sequencing has yet to be fully explored. The MinION sequencer can sequence native DNA and RNA in real-time, providing an opportunity to contrast the utility of DNA and RNA for prediction of drug susceptibility. This study interrogated the genome and transcriptome of four extensively drug-resistant (XDR) K. pneumoniae clinical isolates. The majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kbp). DNA sequencing identified most resistance genes (≥70%) within 2 hours of sequencing. Direct RNA sequencing (with a ∼6x slower pore translocation) was able to identify ≥35% of resistance genes, including aminoglycoside, β-lactam, trimethoprim and sulphonamide and also quinolone, rifampicin, fosfomycin and phenicol in some isolates, within 10 hours of sequencing. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to qRT-PCR across 11 resistance genes. Overall, MinION sequencing rapidly detected the XDR K. pneumoniae resistome and direct RNA sequencing revealed differential expression of these genes.


Author(s):  
Evan E. Ellison ◽  
◽  
James C. Chamness ◽  
Daniel F. Voytas ◽  
◽  
...  

A significant challenge for plant gene editing is the delivery of editing reagents to germline or regenerable cells to recover heritable genetic modifications. Reagent delivery using biolistics or Agrobacterium is only possible with a limited range of species and genotypes, and inefficient editing or lengthy tissue culture steps further limit throughput. Viruses are natural vectors for nucleic acids, and both DNA and RNA plant viruses have been engineered to extend or replace conventional vectors for delivery of gene editing reagents. Here, we review aspects of viral biology essential for engineering vectors, highlight landmark studies using viruses to overcome traditional limitations in gene editing, and outline important considerations for the use of viral vectors in new systems or for new targets. Motivated by fundamental differences in both their infection modes and utility as vectors, DNA and RNA viruses are treated separately.


Virus Genes ◽  
2020 ◽  
Vol 56 (2) ◽  
pp. 236-248 ◽  
Author(s):  
Lumi Viljakainen ◽  
Anna-Maria Borshagovski ◽  
Sami Saarenpää ◽  
Arja Kaitala ◽  
Jaana Jurvansuu

AbstractThe common glow-worms (Lampyris noctiluca) are best known for emission of green light by their larvae and sexually active adult females. However, both their DNA and RNA viruses remain unknown. Glow-worms are virologically interesting, as they are non-social and do not feed as adults, and hence their viral transmission may be limited. We identified viral sequences from 11 different virus taxa by the RNA-sequencing of two Finnish populations of adult glow-worms. The viruses represent nine different virus families and have negative, positive, or double-stranded RNA genomes. We also found a complete retroviral genome. Similar viral sequences were found from the sequencing data of common eastern firefly of North America, a species belonging to the same family (Lampyridae) as that of the common glow-worm. On average, an individual glow-worm had seven different RNA virus types and most of them appeared to establish a stable infection since they were found from glow-worms during two consecutive years. Here we present the characterization of load, prevalence, and interactions for each virus. Most of the glow-worm RNA viruses seem to be transmitted vertically, which may reflect the biology of glow-worms as non-social capital breeders, i.e., they invest stored resources in reproduction.


2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e13509-e13509
Author(s):  
Joseba Rebollo ◽  
Manuel Sureda ◽  
Ramón González-Manzano ◽  
Elena Ma Martínez ◽  
Roman Rostagno ◽  
...  

e13509 Background: Personalized multidrug therapies improve outcomes in patients with drug resistant cancer. For 8 years we have been using microarray-based gene expression profiling (360 procedures) to improve drug selection (Rebollo J et al, 2017; Sureda M et al, 2018). Recently, we have adopted Ion Torrent DNA and RNA Next Generation Sequencing (NGS) to improve the target detection ability. Methods: Since March 2018 DNA (95 analyses) and whole trancriptome RNA (78 analyses) NGS have been performed using Ion Torrent GeneStudio S5 System in fresh-frozen (RNA and DNA sequencing) and paraffin-embedded (DNA sequencing) biopsies obtained from tru-cut or surgical excision procedures from patients with metastatic cancer. Favourable chemotherapy drug profile, treatable (available inhibitory drugs) targets and potentially favourable immunologic signature have been investigated. Results: All biopsies were valid for DNA sequencing but in five patients were invalid (less than 30% viable tumor cells required) for RNA sequencing. Combined DNA and RNA sequencing have been studied in sixty patients with advanced cancer biopsies. Histologies have been NSCLC (12 patents), Breast (9), STS (6), NHL (4), NET (4), Pancreas (4), CRC (4), Ovary/Fallopian tube (3), Prostate (2), Cholangiocarcinoma (2), H&N (2), Esophagus, Bladder, Uterus, Melanoma, SCLC, Anal, Kidney and Cervix (1 each). Most of the patients had been previously treated with systemic therapies. Biopsies have been taken from Lymph nodes (18 patients), Liver (16), Soft Tissues (14), Lung (8), Peritoneum (3) and Adrenal Gland (1). RNA sequencing has predicted favourable chemotherapy drug profile (Median 3 drugs, Range 1-9) in 54 (90%) patients. In addition, treatable targets (Median 3 targets, Range 1-6) have been found in 40 (67%) patients. Favourable immunologic signature has been found in 22 (37%) patients. DNA sequencing has shown treatable (drug available) targets in 14 (23%) patients. In 12 (20%) patients, no targets have been found in any of the sequencing protocols (DNA and RNA). Conclusions: Tumor RNA and DNA sequencing provide potential useful information in personalized cancer treatment decision in a vast majority of advanced cancer patients independent of treatment status.


Molecules ◽  
2019 ◽  
Vol 24 (23) ◽  
pp. 4247 ◽  
Author(s):  
Rita Petrucci ◽  
Isabella Chiarotto ◽  
Leonardo Mattiello ◽  
Daniele Passeri ◽  
Marco Rossi ◽  
...  

Natural methylxanthines, caffeine, theophylline and theobromine, are widespread biologically active alkaloids in human nutrition, found mainly in beverages (coffee, tea, cocoa, energy drinks, etc.). Their detection is thus of extreme importance, and many studies are devoted to this topic. During the last decade, graphene oxide (GO) and reduced graphene oxide (RGO) gained popularity as constituents of sensors (chemical, electrochemical and biosensors) for methylxanthines. The main advantages of GO and RGO with respect to graphene are the easiness and cheapness of synthesis, the notable higher solubility in polar solvents (water, among others), and the higher reactivity towards these targets (mainly due to – interactions); one of the main disadvantages is the lower electrical conductivity, especially when using them in electrochemical sensors. Nonetheless, their use in sensors is becoming more and more common, with the obtainment of very good results in terms of selectivity and sensitivity (up to 5.4 × 10−10 mol L−1 and 1.8 × 10−9 mol L−1 for caffeine and theophylline, respectively). Moreover, the ability of GO to protect DNA and RNA from enzymatic digestion renders it one of the best candidates for biosensors based on these nucleic acids. This is an up-to-date review of the use of GO and RGO in sensors.


Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 628
Author(s):  
Dagmara Baraniak ◽  
Jerzy Boryski

This review covers studies which exploit triazole-modified nucleic acids in the range of chemistry and biology to medicine. The 1,2,3-triazole unit, which is obtained via click chemistry approach, shows valuable and unique properties. For example, it does not occur in nature, constitutes an additional pharmacophore with attractive properties being resistant to hydrolysis and other reactions at physiological pH, exhibits biological activity (i.e., antibacterial, antitumor, and antiviral), and can be considered as a rigid mimetic of amide linkage. Herein, it is presented a whole area of useful artificial compounds, from the clickable monomers and dimers to modified oligonucleotides, in the field of nucleic acids sciences. Such modifications of internucleotide linkages are designed to increase the hybridization binding affinity toward native DNA or RNA, to enhance resistance to nucleases, and to improve ability to penetrate cell membranes. The insertion of an artificial backbone is used for understanding effects of chemically modified oligonucleotides, and their potential usefulness in therapeutic applications. We describe the state-of-the-art knowledge on their implications for synthetic genes and other large modified DNA and RNA constructs including non-coding RNAs.


2021 ◽  
Vol 166 (4) ◽  
pp. 1203-1211
Author(s):  
Caio Bidueira Denani ◽  
Antonio Real-Hohn ◽  
Carlos Alberto Marques de Carvalho ◽  
Andre Marco de Oliveira Gomes ◽  
Rafael Braga Gonçalves

AbstractLactoferrin is part of the innate immune system, with antiviral activity against numerous DNA and RNA viruses. Rhinoviruses, the leading cause of the common cold, are associated with exacerbation of respiratory illnesses such as asthma. Here, we explored the effect of bovine lactoferrin (BLf) on RV-B14 infectivity. Using different assays, we show that the effect of BLf is strongest during adhesion of the virus to the cell and entry. Tracking the internalisation of BLf and virus revealed a degree of colocalisation, although their interaction was only confirmed in vitro using empty viral particles, indicating a possible additional influence of BLf on other infection steps.


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