A rapid metagenomics protocol for the combined detection of RNA viruses, DNA viruses, bacteria and fungi
Ontology highlight
ABSTRACT:
PROVIDER: PRJEB61294 | ENA |
REPOSITORIES: ENA
Dataset's files
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| ERR11635400.fastq.gz | Fastqsanger.gz | |||
| ERR11635500.fastq.gz | Fastqsanger.gz | |||
| ERR11635401.fastq.gz | Fastqsanger.gz | |||
| ERR11635501.fastq.gz | Fastqsanger.gz | |||
| ERR11635402.fastq.gz | Fastqsanger.gz |
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Project description:SDL-TOPO protocol was applied for the detection of lagging-strand in replicating budding yeast strain. Genomic DNA was extracted from cdc9 mutant strain, and served for library preparation with SDL-TOPO. To see the sensitivity of SDL-TOPO for the detection of lagging strand, various amounts of genomic DNA were used for the library preparation: 75 ng, 25 ng, 7.5 ng, 2.5 ng, 750 pg, 250 pg, 75 pg, and 25 pg. The prepared libraries were sequenced on Illumina iSeq100 with paired-end mode of 150x2. Only read 1 was served for mapping with bowtie2 against reference sequence sacSer3. The mapped reads were separated strand specifically, and read depths were visualized. The strand-biased mapping patterens were observed for libraries prepared from each amount of input DNA.
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Project description:BackgroundEndophthalmitis is an ophthalmologic emergency requiring accurate and rapid diagnosis for treatment. Currently, the diagnosis commonly relies on culture and molecular biology, which falls short of clinical rapid diagnosis. The purpose of this study was to evaluate the feasibility of a self-build Microfluidic Real-time Polymerase Chain Reaction (RT-PCR) System for rapidly identifying potential pathogens of endophthalmitis.MethodsThis study included 22 patients who presented to Shenzhen Eye Hospital and the Ophthalmology Department of the Affiliated Hospital of Guizhou Medical University in China between January 2023 and March 2024. The samples were cultured using conventional methods and underwent Microfluidic RT-PCR and metagenomic next-generation sequencing (mNGS).ResultsThe Microfluidic RT-PCR System identified pathogens in 11 of 22 cases (50.00%), compared with 40.91% for microbiology culture. 14 cases (63.64%) had concordant results, and 5 cases were positive for the microfluidic system only. The agreements between culture and microfluidic system, as well as culture and mNGS were 100.00% (6/6) and 50.00% (3/6), respectively. The average waiting time for the microfluidic system was about 30 min if excepting DNA extraction time, which was much shorter than 2.88 days for culture and 1.57 days for mNGS.ConclusionThe microfluidic-based RT-PCR system was preliminarily proved to be a sensitive, easy-to-operate, and rapid in-hospital technology. It is expected to become a rapid diagnostic platform for endophthalmitis.
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Project description:BackgroundThe International Space Station (ISS) is a unique and complex built environment with the ISS surface microbiome originating from crew and cargo or from life support recirculation in an almost entirely closed system. The Microbial Tracking 1 (MT-1) project was the first ISS environmental surface study to report on the metagenome profiles without using whole-genome amplification. The study surveyed the microbial communities from eight surfaces over a 14-month period. The Microbial Tracking 2 (MT-2) project aimed to continue the work of MT-1, sampling an additional four flights from the same locations, over another 14 months.MethodsEight surfaces across the ISS were sampled with sterile wipes and processed upon return to Earth. DNA extracted from the processed samples (and controls) were treated with propidium monoazide (PMA) to detect intact/viable cells or left untreated and to detect the total DNA population (free DNA/compromised cells/intact cells/viable cells). DNA extracted from PMA-treated and untreated samples were analyzed using shotgun metagenomics. Samples were cultured for bacteria and fungi to supplement the above results.ResultsStaphylococcus sp. and Malassezia sp. were the most represented bacterial and fungal species, respectively, on the ISS. Overall, the ISS surface microbiome was dominated by organisms associated with the human skin. Multi-dimensional scaling and differential abundance analysis showed significant temporal changes in the microbial population but no spatial differences. The ISS antimicrobial resistance gene profiles were however more stable over time, with no differences over the 5-year span of the MT-1 and MT-2 studies. Twenty-nine antimicrobial resistance genes were detected across all samples, with macrolide/lincosamide/streptogramin resistance being the most widespread. Metagenomic assembled genomes were reconstructed from the dataset, resulting in 82 MAGs. Functional assessment of the collective MAGs showed a propensity for amino acid utilization over carbohydrate metabolism. Co-occurrence analyses showed strong associations between bacterial and fungal genera. Culture analysis showed the microbial load to be on average 3.0 × 105 cfu/m2 CONCLUSIONS: Utilizing various metagenomics analyses and culture methods, we provided a comprehensive analysis of the ISS surface microbiome, showing microbial burden, bacterial and fungal species prevalence, changes in the microbiome, and resistome over time and space, as well as the functional capabilities and microbial interactions of this unique built microbiome. Data from this study may help to inform policies for future space missions to ensure an ISS surface microbiome that promotes astronaut health and spacecraft integrity. Video Abstract.
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