scholarly journals A metagenomic DNA sequencing assay that is robust against environmental DNA contamination

2021 ◽  
Author(s):  
Omary Mzava ◽  
Alexandre Pellan Cheng ◽  
Adrienne Chang ◽  
Sami Smalling ◽  
Liz-Audrey Djomnang Kounatse ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Dna Isolation ◽  
Environmental Dna ◽  
Metagenomic Sequencing ◽  
Metagenomic Dna ◽  
Dna Contamination ◽  
Urinary Microbiome ◽  
Inflammatory Bowel ◽  
Core Idea ◽  
Microbial Dna

Metagenomic DNA sequencing is a powerful tool to characterize microbial communities but is sensitive to environmental DNA contamination, in particular when applied to samples with low microbial biomass. Here, we present contamination-free metagenomic DNA sequencing (Coffee-seq), a metagenomic sequencing assay that is robust against environmental contamination. The core idea of Coffee-seq is to tag the DNA in the sample prior to DNA isolation and library preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. We applied Coffee-seq to screen for infections from microorganisms with low burden in blood and urine, to identify COVID-19 co-infection, to characterize the urinary microbiome, and to identify microbial DNA signatures of inflammatory bowel disease in blood.

scholarly journals Discriminating between JCPyV and BKPyV in Urinary Virome Data Sets

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1041
Author(s):  
Rita Mormando ◽  
Alan J. Wolfe ◽  
Catherine Putonti
Keyword(s):  
Jc Virus ◽  
Sequence Similarity ◽  
Bk Virus ◽  
Data Sets ◽  
Metagenomic Sequencing ◽  
Significant Sequence Similarity ◽  
Sequence Identity ◽  
Shotgun Metagenomic Sequencing ◽  
Urinary Microbiome ◽  
Six Genes

Polyomaviruses are abundant in the human body. The polyomaviruses JC virus (JCPyV) and BK virus (BKPyV) are common viruses in the human urinary tract. Prior studies have estimated that JCPyV infects between 20 and 80% of adults and that BKPyV infects between 65 and 90% of individuals by age 10. However, these two viruses encode for the same six genes and share 75% nucleotide sequence identity across their genomes. While prior urinary virome studies have repeatedly reported the presence of JCPyV, we were interested in seeing how JCPyV prevalence compares to BKPyV. We retrieved all publicly available shotgun metagenomic sequencing reads from urinary microbiome and virome studies (n = 165). While one third of the data sets produced hits to JCPyV, upon further investigation were we able to determine that the majority of these were in fact BKPyV. This distinction was made by specifically mining for JCPyV and BKPyV and considering uniform coverage across the genome. This approach provides confidence in taxon calls, even between closely related viruses with significant sequence similarity.

scholarly journals Gut microbiota differs between children with Inflammatory Bowel Disease and healthy siblings in taxonomic and functional composition: a metagenomic analysis

2017 ◽  
Vol 312 (4) ◽  
pp. G327-G339 ◽  
Author(s):  
Rebecca L. Knoll ◽  
Kristoffer Forslund ◽  
Jens Roat Kultima ◽  
Claudius U. Meyer ◽  
Ulrike Kullmer ◽  
...  
Keyword(s):  
Inflammatory Bowel Disease ◽  
Gut Microbiota ◽  
Bowel Disease ◽  
Virulence Genes ◽  
Metagenomic Sequencing ◽  
Fecal Microbiome ◽  
Microbial Dysbiosis ◽  
Healthy Siblings ◽  
Inflammatory Bowel ◽  
Sibling Study

Current treatment for pediatric inflammatory bowel disease (IBD) patients is often ineffective, with serious side effects. Manipulating the gut microbiota via fecal microbiota transplantation (FMT) is an emerging treatment approach but remains controversial. We aimed to assess the composition of the fecal microbiome through a comparison of pediatric IBD patients to their healthy siblings, evaluating risks and prospects for FMT in this setting. A case-control (sibling) study was conducted analyzing fecal samples of six children with Crohn’s disease (CD), six children with ulcerative colitis (UC) and 12 healthy siblings by metagenomic sequencing. In addition, lifetime antibiotic intake was retrospectively determined. Species richness and diversity were significantly reduced in UC patients compared with control [Mann-Whitney U-test false discovery rate (MWU FDR) = 0.011]. In UC, bacteria positively influencing gut homeostasis, e.g., Eubacterium rectale and Faecalibacterium prausnitzii, were significantly reduced in abundance (MWU FDR = 0.05). Known pathobionts like Escherichia coli were enriched in UC patients (MWU FDR = 0.084). Moreover, E. coli abundance correlated positively with that of several virulence genes (SCC > 0.65, FDR < 0.1). A shift toward antibiotic-resistant taxa in both IBD groups distinguished them from controls [MWU Benjamini-Hochberg-Yekutieli procedure (BY) FDR = 0.062 in UC, MWU BY FDR = 0.019 in CD). The collected results confirm a microbial dysbiosis in pediatric UC, and to a lesser extent in CD patients, replicating associations found previously using different methods. Taken together, these observations suggest microbiotal remodeling therapy from family donors, at least for children with UC, as a viable option. NEW & NOTEWORTHY In this sibling study, prior reports of microbial dysbiosis in IBD patients from 16S rRNA sequencing was verified using deep shotgun sequencing and augmented with insights into the abundance of bacterial virulence genes and bacterial antibiotic resistance determinants, seen against the background of data on the specific antibiotic intake of each of the study participants. The observed dysbiosis, which distinguishes patients from siblings, highlights such siblings as potential donors for microbiotal remodeling therapy in IBD.

scholarly journals Molecular diagnosis of orthopaedic device infection direct from sonication fluid by metagenomic sequencing

2017 ◽  
Author(s):  
Teresa L. Street ◽  
Nicholas D. Sanderson ◽  
Bridget L. Atkins ◽  
Andrew J. Brent ◽  
Kevin Cole ◽  
...  
Keyword(s):  
Diagnostic Information ◽  
Species Level ◽  
Metagenomic Sequencing ◽  
Tissue Samples ◽  
Bacterial Dna ◽  
Dna Contamination ◽  
Prosthetic Joint ◽  
Device Infection ◽  
Fluid Culture ◽  
Orthopaedic Device

AbstractCulture of multiple periprosthetic tissue samples is the current gold-standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through sonication fluid culture of explants. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of complete bacterial DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI.We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopaedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 samples (derivation set), we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and validated our findings in 47 independent samples.Compared to sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69(88%,95%CI 77-94%) (derivation samples 35/38[92%,79-98%]; validation 26/31[84%,66-95%]), and genus-level sensitivity was 64/69(93%,84-98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97(88%,79-93%) (derivation 43/50[86%,73-94%], validation 42/47[89%,77-96%]). High levels of human DNA contamination were seen despite use of laboratory methods to remove it. Rigorous laboratory good practice was required to prevent bacterial DNA contamination.We demonstrate metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings combined with increasing availability of portable, random-access sequencing technology offers the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI.

scholarly journals A Simple Technique for the Identification of Environmental DNA (eDNA ) in the Water Samples

2021 ◽  
pp. 77-80
Author(s):  
Chitra K. Y.
Keyword(s):  
Water Samples ◽  
Large Scale ◽  
Dna Isolation ◽  
Simple Technique ◽  
Environmental Dna ◽  
Water Bodies ◽  
Easy Method ◽  
Selective Staining ◽  
Generation Sequencing ◽  
Dark Pink

The environmental DNA(eDNA) is the DNA that is shed by the organisms in their environment by different ways viz. , mucous, faeces, skin, eggs, sperms and also when these organisms die due to natural death or disease. The eDNA will persist for several days. Identification of eDNA is a useful method of determining the organisms present in an aquatic environment like amphibians, reptiles, fishes , insects and larval forms of some of these organisms. By analysing the e-DNA it is possible to monitor the species distribution in water bodies like lakes and ponds simply by collecting a sample of water. The technique can be applied for the survey of the water bodies on a large scale for the genomic, taxonomic as well as pollutional studies. The DNA isolation procedures that are available are laborious and time consuming. Therefore, during the present study, a simplified method was devised i. e. , isolation of eDNA with ethanol after which Feulgen stain was applied to identify and confirm it, as it is an easy method before proceeding to work with the isolated eDNA using other techniqnies for further studies. The Feulgen method is used for the selective staining and the localisation of the DNA in the tissues but is adopted during the present study for the water samples for quick identification of eDNA. The smear of eDNA stained with Feulgen showed dark pink or magenta colour under the microscope where it was concentrated but stained lightly when dispersed and fragmented as observed in the present study. Further studies of the isolated eDNA are in progress in our laboratory for quantifying and sequencing eDNA using latest techniques like next generation sequencing for the identification of fish species in the lakes.

scholarly journals Influence of DNA extraction kits on the fungal DNA metabarcoding for freshwater environmental DNA

2021 ◽  
Author(s):  
Shunsuke Matsuoka ◽  
Yoriko Sugiyama ◽  
Mariko Nagano ◽  
Hideyuki Doi
Keyword(s):  
Dna Extraction ◽  
Environmental Dna ◽  
Operational Taxonomic Unit ◽  
Pcr Inhibition ◽  
Dna Metabarcoding ◽  
Fungal Dna ◽  
Diversity Studies ◽  
Microbial Dna ◽  
Methodological Studies ◽  
Biodiversity Analysis

Background: Environmental DNA (eDNA) metabarcoding is a rapidly expanding technique for efficient biodiversity monitoring, especially of animals. Recently, the usefulness of aquatic eDNA in monitoring the diversity of both terrestrial and aquatic fungi has been suggested. In eDNA studies, different experimental factors, such as DNA extraction kits or methods, can affect the subsequent analyses and the results of DNA metabarcoding. However, few methodological studies have been carried out on eDNA of fungi, and little is known about how experimental procedures can affect the results of biodiversity analysis. In this study, we focused on the effect of the DNA extraction method on fungal DNA metabarcoding using freshwater samples obtained from rivers and lakes. Methods: DNA was extracted from freshwater samples using the DNeasy PowerSoil kit, which is mainly used to extract microbial DNA from soil, and the DNeasy Blood & Tissue kit, which is commonly used for eDNA studies on animals. We then compared PCR inhibition and fungal DNA metabarcoding results [i.e., operational taxonomic unit (OTU) number and composition] of the extracted samples. Results: No PCR inhibition was detected in any of the samples, and no significant differences in the number of OTUs and OTU compositions were detected between the samples processed using different kits. These results indicate that both DNA extraction kits may provide similar diversity results for the river and lake samples evaluated in this study. Therefore, it may be possible to evaluate the diversity of fungi using a unified experimental method, even with samples obtained for diversity studies on other taxa such as those of animals.

Extracellular DNA in environmental samples: Occurrence, extraction, quantification, and impact on microbial biodiversity assessment

2021 ◽  
Author(s):  
Sakcham Bairoliya ◽  
Jonas Koh Zhi Xiang ◽  
Bin Cao
Keyword(s):  
Dna Sequencing ◽  
Microbial Communities ◽  
Dna Extraction ◽  
Environmental Samples ◽  
Environmental Dna ◽  
Extracellular Dna ◽  
Biodiversity Assessment ◽  
Microbial Biodiversity ◽  
Technical Details ◽  
The Impact

Environmental DNA, i.e., DNA directly extracted from environmental samples, has been applied to understand microbial communities in the environments and to monitor contemporary biodiversity in the conservation context. Environmental DNA often contains both intracellular DNA (iDNA) and extracellular DNA (eDNA). eDNA can persist in the environment and complicate environmental DNA sequencing-based analyses of microbial communities and biodiversity. Although several studies acknowledged the impact of eDNA on DNA-based profiling of environmental communities, eDNA is still being neglected or ignored in most studies dealing with environmental samples. In this article, we summarize key findings on eDNA in environmental samples and discuss the methods used to extract and quantify eDNA as well as the importance of eDNA on the interpretation of experimental results. We then suggest several factors to consider when designing experiments and analyzing data to negate or determine the contribution of eDNA to environmental DNA-based community analyses. This field of research will be driven forward by: (i) carefully designing environmental DNA extraction pipelines by taking into consideration technical details in methods for eDNA extraction/removal and membrane-based filtration and concentration; (ii) quantifying eDNA in extracted environmental DNA using multiple methods including qPCR and fluorescent DNA binding dyes; (iii) carefully interpretating effect of eDNA on DNA-based community analyses at different taxonomic levels; and (iv) when possible, removing eDNA from environmental samples for DNA-based community analyses.

scholarly journals Unusual Microbial Xylanases from Insect Guts

2004 ◽  
Vol 70 (6) ◽  
pp. 3609-3617 ◽  
Author(s):  
YaLi Brennan ◽  
Walter N. Callen ◽  
Leif Christoffersen ◽  
Paul Dupree ◽  
Florence Goubet ◽  
...  
Keyword(s):  
Recombinant Dna ◽  
Xylanase Activity ◽  
Environmental Dna ◽  
Functional Assay ◽  
Genomic Libraries ◽  
Dna Libraries ◽  
Polymeric Substrates ◽  
Microbial Dna ◽  
Hydrolysis Of ◽  
Uncultured Microorganisms

ABSTRACT Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted β-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.

scholarly journals P053 Overabundance of Lactobacillus species in gut microbiota of IBD patients

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S160-S161
Author(s):  
D Khusnutdinova ◽  
M Markelova ◽  
M Siniagina ◽  
E Boulygina ◽  
S Abdulkhakov ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Intestinal Microbiota ◽  
Active Phase ◽  
Bioinformatic Analysis ◽  
Lactobacillus Species ◽  
Control Group ◽  
Metagenomic Sequencing ◽  
Shotgun Metagenomic Sequencing ◽  
Inflammatory Bowel ◽  
Stool Samples

Abstract Background Changes in the composition of gut microbiota, and their metabolic pathways, are important factors in the pathogenesis of inflammatory bowel disease (IBD). Many clinical trials have shown that taking probiotics based on Lactobacillus has a positive effect on patients with IBD. However, Lactobacillus should be used more carefully during the active phase of IBD, since some strains can negatively affect the pathogenesis of the disease1,2. The aim of this study was to assess the diversity of Lactobacillus species in the gut microbiome of IBD patients and healthy volunteers. Methods In the study, 62 stool samples from healthy people, 31 from patients with Crohn’s disease (CD), and 34 - ulcerative colitis (UC) in active phase were analyzed. DNA was isolated using the QIAamp Fast DNA Stool Mini Kit (Qiagen, USA) following with shotgun metagenomic sequencing the NextSeq 500 (project #0671-2020-0058). Bioinformatic analysis was performed with the MetaPhlAn2 package. Results An increased relative abundance of Lactobacillus was found in patients with IBD (3.2% ± 6.6% in CD and 1.6% ± 2.8 in UC) compared to healthy individuals (0.3% ± 1.2%, p&lt;0.05). In the control group, Lactobacillus were absent in 41% of samples and 1–5 species were found in 58% of samples. Most CD and UC patients are characterized by the presence of 3 to 5 species of Lactobacillus (38% and 31%, respectively). For 23% of CD patients and 26% of UC patients, 6 to 9 types of Lactobacillus were found. Some patients with IBD have more than 10 different types of Lactobacillus in the gut microbiota (Fig.1). The intestinal microbiota in IBD patients is characterized by an increased abundance of several species: L. salivarius, L. gasseri, L. mucosae, as well as L. casei paracasei in patients with CD and L. vaginalis in patients with UC (Fig.2). Conclusion The composition of the intestinal microbiota of IBD patients differs significantly in terms of Lactobacillus proportion and species diversity. Overabundance of five Lactobacillus species could be associated with the active phase of IBD. References

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