Investigating Extracellular DNA Release in Staphylococcus xylosus Biofilm In Vitro

Staphylococcus xylosus forms biofilm embedded in an extracellular polymeric matrix. As extracellular DNA (eDNA) resulting from cell lysis has been found in several staphylococcal biofilms, we investigated S. xylosus biofilm in vitro by a microscopic approach and identified the mechanisms involved in cell lysis by a transcriptomic approach. Confocal laser scanning microscopy (CLSM) analyses of the biofilms, together with DNA staining and DNase treatment, revealed that eDNA constituted an important component of the matrix. This eDNA resulted from cell lysis by two mechanisms, overexpression of phage-related genes and of cidABC encoding a holin protein that is an effector of murein hydrolase activity. This lysis might furnish nutrients for the remaining cells as highlighted by genes overexpressed in nucleotide salvage, in amino sugar catabolism and in inorganic ion transports. Several genes involved in DNA/RNA repair and genes encoding proteases and chaperones involved in protein turnover were up-regulated. Furthermore, S. xylosus perceived osmotic and oxidative stresses and responded by up-regulating genes involved in osmoprotectant synthesis and in detoxification. This study provides new insight into the physiology of S. xylosus in biofilm.

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

Although 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.

DNase Treatment Improves Viral Enrichment in Agricultural Soil Viromes

Viromics is becoming an increasingly popular method for characterizing soil viral communities. DNase treatment of the viral size fraction prior to DNA extraction is meant to reduce contaminating free DNA and is a common step within viromics protocols to ensure that sequences are of viral origin.

Dual RNA-Seq Enables Full-Genome Assembly of Measles Virus and Characterization of Host–Pathogen Interactions

Measles virus (MeV) has a negative-sense 15 kb long RNA genome, which is generally conserved. Recent advances in high-throughput sequencing (HTS) and Dual RNA-seq allow the analysis of viral RNA genomes and the discovery of viral infection biomarkers, via the simultaneous characterization of the host transcriptome. However, these host–pathogen interactions remain largely unexplored in MeV infections. We performed untargeted Dual RNA-seq in 6 pharyngeal and 6 peripheral blood mononuclear cell (PBMCs) specimens from patients with MeV infection, as confirmed via routine real-time PCR testing. Following optimised DNase treatment of total nucleic acids, we used the pharyngeal samples to build poly-A-enriched NGS libraries. We reconstructed the viral genomes using the pharyngeal datasets and we further conducted differential expression, gene-ontology and pathways enrichment analysis to compare both the pharyngeal and the peripheral blood transcriptomes of the MeV-infected patients vs. control groups of healthy individuals. We obtained 6 MeV genotype-B3 full-genome sequences. We minutely analyzed the transcriptome of the MeV-infected pharyngeal epithelium, detecting all known viral infection biomarkers, but also revealing a functional cluster of local antiviral and inflammatory immune responses, which differ substantially from those observed in the PBMCs transcriptome. The application of Dual RNA-seq technologies in MeV-infected patients can potentially provide valuable information on the virus genome structure and the cellular innate immune responses and drive the discovery of new targets for antiviral therapy.

The Effect of Genomic DNA Contamination on the Detection of Circulating Long Non-Coding RNAs: The Paradigm of MALAT1

The presence of contaminating gDNA in RNA preparations is a frequent cause of false positives in RT-PCR-based analysis. However, in some cases, this cannot be avoided, especially when there are no exons–intron junctions in the lncRNA sequences. Due to the lack of exons in few of long noncoding RNAs (lncRNAs) and the lack of DNAse treatment step in most studies reported so far, serious questions are raised about the specificity of lncRNA detection and the potential of reporting false-positive results. We hypothesized that minute amounts of gDNA usually co-extracted with RNA could give false-positive signals since primers would specifically bind to gDNA due to the lack of junction. In the current study, we evaluated the effect of gDNA and other forms of DNA like extrachromosomal circular DNAs (eccDNAs) contamination and the importance of including a DNAse treatment step on lncRNAsexpression.As a model, we have chosen as one of the most widely studied lncRNAs in cancer namely MALAT1, which lacks exons. When we tested this hypothesis in plasma and primary tissue samples from NSCLC patients, our findings clearly indicated that results on MALAT1 expression are highly affected by the presence of DNA contamination and that the DNAse treatment step is absolutely necessary to avoid false positive results.

Development of a method for the determination of the JAK2 gene mRNA in venous blood and assessment of its diagnostic value in oncohematology

Overactive JAK pathway signaling is a hallmark of immune diseases and critically affects on inflammation and coagulation. A number of mutations in the JAK2 gene act as driving forces of myeloproliferative neoplasms (MPN), the pathogenesis of certain variants of acute leukemia, a number of solid malignancies and cardiovascular diseases. Assays for quantifying JAK2 mRNA in circulating blood cells can be used as a marker associated with the activity of this enzyme. Development of an original method for detecting JAK2 mRNA in venous blood and assessment of the possible diagnostic value in chronic oncohematological diseases. The development of an RT-PCR method for determining the expression of the JAK2 gene mRNA in venous blood samples was carried out in accordance with the MIQE requirements. Primers and TaqMan probes were designed using the Primer3 program, taking into account the possibility of excluding subsequent DNase treatment. The stability of the investigated mRNA was assessed in vacutainers with different anticoagulants and depending on the storage time of the samples. The study of the expression of JAK2 mRNA in blood leukocytes of 41 patients with B-CLL, 16 patients with CML, 12 patients with multiple myeloma and 39 donors using the developed “real-time” PCR method. The study revealed a decrease in the level of JAK2 mRNA in venous blood samples in patients with primary CLL, but not with CML or with multiple myeloma. The level of the marker in the majority of patients with CLL after the start of therapy returned to the range typical for healthy people. It has been shown that the values of the relative expression of JAK2 mRNA are most stable in the range of 2 - 7 hours after taking blood in a vacutainer with EDTA. An original RT-PCR method was developed for the quantitative determination of JAK2 mRNA in venous blood samples, which meets the requirements of the MIQE system. Determination of JAK2 mRNA can be useful for clarifying the pathogenesis features of certain diseases involving impaired Janus kinase activity and can become a promising marker for prognosis and assessment of the effectiveness of therapy.

DNase treatment improves viral enrichment in agricultural soil viromes

The small genomes of most viruses make it difficult to fully capture viral diversity in metagenomes dominated by DNA from cellular organisms. Viral size-fraction metagenomics (viromics) protocols facilitate enrichment of viral DNA from environmental samples, and these protocols typically include a DNase treatment of the post-0.2 μm viromic fraction to remove contaminating free DNA prior to virion lysis. However, DNase may also remove desirable viral genomic DNA (e.g., contained in virions compromised due to frozen storage or laboratory processing), suggesting that DNase-untreated viromes might be useful in some cases. In order to understand how virome preparation with and without DNase treatment influences the resultant data, here we compared 15 soil viromes (7 DNase-treated, 8 untreated) from 8 samples collected from agricultural fields prior to tomato planting. DNase-treated viromes yielded significantly more assembled viral contigs, contained significantly less non-viral microbial DNA, and recovered more viral populations (vOTUs) through read mapping. However, DNase-treated and untreated viromes were statistically indistinguishable, in terms of ecological patterns across viral communities. Although results suggest that DNase treatment is preferable where possible, in comparison to previously reported total metagenomes from the same samples, both DNase-treated and untreated viromes were significantly enriched in viral signatures by all metrics compared, including a ~225 times greater proportion of viral reads in untreated viromes compared to total metagenomes. Thus, even without DNase treatment, viromics was preferable to total metagenomics for capturing viral diversity in these soils, suggesting that preparation of DNase-untreated viromes can be worthwhile when DNase treatment is not possible

Enrichment approach for unbiased sequencing of respiratory syncytial virus directly from clinical samples

Background: Nasopharyngeal samples contain higher quantities of bacterial and host nucleic acids relative to viruses; presenting challenges during virus metagenomics sequencing, which underpins agnostic sequencing protocols. We aimed to develop a viral enrichment protocol for unbiased whole-genome sequencing of respiratory syncytial virus (RSV) from nasopharyngeal samples using the Oxford Nanopore Technology (ONT) MinION platform. Methods: We assessed two protocols using RSV positive samples. Protocol 1 involved physical pre-treatment of samples by centrifugal processing before RNA extraction, while Protocol 2 entailed direct RNA extraction without prior enrichment. Concentrates from Protocol 1 and RNA extracts from Protocol 2 were each divided into two fractions; one was DNase treated while the other was not. RNA was then extracted from both concentrate fractions per sample and RNA from both protocols converted to cDNA, which was then amplified using the tagged Endoh primers through Sequence-Independent Single-Primer Amplification (SISPA) approach, a library prepared, and sequencing done. Statistical significance during analysis was tested using the Wilcoxon signed-rank test. Results: DNase-treated fractions from both protocols recorded significantly reduced host and bacterial contamination unlike the untreated fractions (in each protocol p<0.01). Additionally, DNase treatment after RNA extraction (Protocol 2) enhanced host and bacterial read reduction compared to when done before (Protocol 1). However, neither protocol yielded whole RSV genomes. Sequenced reads mapped to parts of the nucleoprotein (N gene) and polymerase complex (L gene) from Protocol 1 and 2, respectively. Conclusions: DNase treatment was most effective in reducing host and bacterial contamination, but its effectiveness improved if done after RNA extraction than before. We attribute the incomplete genome segments to amplification biases resulting from the use of short length random sequence (6 bases) in tagged Endoh primers. Increasing the length of the random nucleotides from six hexamers to nine or 12 in future studies may reduce the coverage biases.