scholarly journals Identifying Taxonomic Units in Metagenomic DNA Streams

2020 ◽  
Author(s):  
Vicky Zheng ◽  
Ahmet Erdem Sariyuce ◽  
Jaroslaw Zola
Keyword(s):  
Real Time ◽  
Dna Analysis ◽  
Metagenomic Data ◽  
Connected Components ◽  
Redundant Information ◽  
Metagenomic Dna ◽  
Operational Taxonomic Units ◽  
Oxford Nanopore ◽  
Overlap Graphs ◽  
Memory Efficient

AbstractWith the emergence of portable DNA sequencers, such as Oxford Nanopore Technology MinION, metagenomic DNA sequencing can be performed in real-time and directly in the field. However, because metagenomic DNA analysis is computationally and memory intensive, and the current methods are designed for batch processing, the current metagenomic tools are not well suited for mobile devices.In this paper, we propose a new memory-efficient method to identify Operational Taxonomic Units (OTUs) in metagenomic DNA streams. Our method is based on finding connected components in overlap graphs constructed over a real-time stream of long DNA reads as produced by MinION platform. We propose an efficient algorithm to maintain connected components when an overlap graph is streamed, and show how redundant information can be removed from the stream by transitive closures. Through experiments on simulated and real-world metagenomic data, we demonstrate that the resulting solution is able to recover OTUs with high precision while remaining suitable for mobile computing devices.

scholarly journals Haemophilus influenzae Meningitis Direct Diagnosis by Metagenomic Next-Generation Sequencing: A Case Report

Pathogens ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 461
Author(s):  
Madjid Morsli ◽  
Quentin Kerharo ◽  
Jeremy Delerce ◽  
Pierre-Hugues Roche ◽  
Lucas Troude ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Haemophilus Influenzae ◽  
Real Time ◽  
Real Time Pcr ◽  
Point Of Care ◽  
Next Generation ◽  
Oxford Nanopore ◽  
Sequence Encoding ◽  
Oxford Nanopore Technologies ◽  
Generation Sequencing

Current routine real-time PCR methods used for the point-of-care diagnosis of infectious meningitis do not allow for one-shot genotyping of the pathogen, as in the case of deadly Haemophilus influenzae meningitis. Real-time PCR diagnosed H. influenzae meningitis in a 22-year-old male patient, during his hospitalisation following a more than six-metre fall. Using an Oxford Nanopore Technologies real-time sequencing run in parallel to real-time PCR, we detected the H. influenzae genome directly from the cerebrospinal fluid sample in six hours. Furthermore, BLAST analysis of the sequence encoding for a partial DUF417 domain-containing protein diagnosed a non-b serotype, non-typeable H.influenzae belonging to lineage H. influenzae 22.1-21. The Oxford Nanopore metagenomic next-generation sequencing approach could be considered for the point-of-care diagnosis of infectious meningitis, by direct identification of pathogenic genomes and their genotypes/serotypes.

scholarly journals Integrated Extreme Real-Time PCR and High-Speed Melting Analysis in 52 to 87 Seconds

2019 ◽  
Vol 65 (2) ◽  
pp. 263-271 ◽  
Author(s):  
Joseph T Myrick ◽  
Robert J Pryor ◽  
Robert A Palais ◽  
Sean J Ison ◽  
Lindsay Sanford ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Speed ◽  
Dna Analysis ◽  
Point Of Care ◽  
Turnaround Time ◽  
Single Nucleotide Variants ◽  
Cycle Times ◽  
Pcr Products ◽  
Melting Analysis

Abstract BACKGROUND Extreme PCR in <30 s and high-speed melting of PCR products in <5 s are recent advances in the turnaround time of DNA analysis. Previously, these steps had been performed on different specialized instruments. Integration of both extreme PCR and high-speed melting with real-time fluorescence monitoring for detection and genotyping is presented here. METHODS A microfluidic platform was enhanced for speed using cycle times as fast as 1.05 s between 66.4 °C and 93.7 °C, with end point melting rates of 8 °C/s. Primer and polymerase concentrations were increased to allow short cycle times. Synthetic sequences were used to amplify fragments of hepatitis B virus (70 bp) and Clostridium difficile (83 bp) by real-time PCR and high-speed melting on the same instrument. A blinded genotyping study of 30 human genomic samples at F2 c.*97, F5 c.1601, MTHFR c.665, and MTHFR c.1286 was also performed. RESULTS Standard rapid-cycle PCR chemistry did not produce any product when total cycling times were reduced to <1 min. However, efficient amplification was possible with increased primer (5 μmol/L) and polymerase (0.45 U/μL) concentrations. Infectious targets were amplified and identified in 52 to 71 s. Real-time PCR and genotyping of single-nucleotide variants from human DNA was achieved in 75 to 87 s and was 100% concordant to known genotypes. CONCLUSIONS Extreme PCR with high-speed melting can be performed in about 1 min. The integration of extreme PCR and high-speed melting shows that future molecular assays at the point of care for identification, quantification, and variant typing are feasible.

scholarly journals Metagenomic and satellite analyses of red snow in the Russian Arctic

PeerJ ◽  
2015 ◽  
Vol 3 ◽  
pp. e1491 ◽  
Author(s):  
Nao Hisakawa ◽  
Steven D. Quistad ◽  
Eric R. Hester ◽  
Daria Martynova ◽  
Heather Maughan ◽  
...  
Keyword(s):  
Image Analysis ◽  
Dna Analysis ◽  
Satellite Image ◽  
Polar Regions ◽  
Russian Arctic ◽  
Metagenomic Dna ◽  
Satellite Imaging ◽  
Franz Josef Land ◽  
Satellite Image Analysis ◽  
Snow And Ice

Cryophilic algae thrive in liquid water within snow and ice in alpine and polar regions worldwide. Blooms of these algae lower albedo (reflection of sunlight), thereby altering melting patterns (Kohshima, Seko & Yoshimura, 1993; Lutz et al., 2014; Thomas & Duval, 1995). Here metagenomic DNA analysis and satellite imaging were used to investigate red snow in Franz Josef Land in the Russian Arctic. Franz Josef Land red snow metagenomes confirmed that the communities are composed of the autotrophChlamydomonas nivalisthat is supporting a complex viral and heterotrophic bacterial community. Comparisons with white snow communities from other sites suggest that white snow and ice are initially colonized by fungal-dominated communities and then succeeded by the more complexC. nivalis-heterotroph red snow. Satellite image analysis showed that red snow covers up to 80% of the surface of snow and ice fields in Franz Josef Land and globally. Together these results show thatC. nivalissupports a local food web that is on the rise as temperatures warm, with potential widespread impacts on alpine and polar environments worldwide.

PSXIV-24 Real-time, on-site whole genome sequencing with oxford nanopore technologies’ MinION

2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 257-258
Author(s):  
Hanna Ostrovski ◽  
Rodrigo Pelicioni Savegnago ◽  
Wen Huang ◽  
Cedric Gondro
Keyword(s):  
Real Time ◽  
Ease Of Use ◽  
Whole Genome Sequence ◽  
Genomic Sequencing ◽  
Whole Genome ◽  
Illumina Hiseq ◽  
Base Calling ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

Abstract Most quantitative geneticists are traditionally trained for data analysis in genetic evaluation and genomic prediction, but rarely have extensive knowledge of molecular genetics or experience in experimental labs. Recent products, such as those launched by Oxford Nanopore Technologies (ONT), give those quantitative geneticists a comprehensible and hands-on toolkit to explore DNA sequencing. The ‘MinION’, a small DNA sequencer, is of interest for quantitative geneticists due to both the minimal learning curve and the non-proprietary USB connectivity. This device is small enough to be portable, allowing for potential real-time, on-farm sequencing. The objective of this project is to compare the whole genome sequence (WGS) output of the MinION sequencer to that of the Illumina HiSeq 4000. Blood was collected from a 6-month-old Akaushi calf born on a Michigan State University farm. DNA was extracted from the sample using the QIAamp DNA Blood Kit from Qiagen, and library DNA ligation preparation (SQK-LSK109) from ONT was used. After base-calling with guppy software (provided by ONT), the data were preprocessed and experimental runs with the MinION were compared using quality control. Finally, the data were aligned with guppy software, and was compared to the aligned WGS obtained with Illumina HiSeq. Quality results from each MinION indicate that, despite the low amount of sequence collected in each run (~225,303 reads per run), the quality of bases sequenced was high (Q≥7). The aligned data from the Illumina sequencer provided 40x coverage of the genome, with a total of 739,339,742 reads. Although the amount of data obtained with MinION is much smaller than that of Illumina HiSeq, the high quality of MinION’s data combined with its ease of use give an opportunity of genomic sequencing for users who are either inexperienced or do not have access to large genomic sequencing devices.

Real-Time Point Feature Extraction Based on Connected Components Labeling and Distributed Computing

2015 ◽  
Vol 35 (2) ◽  
pp. 0210001
Author(s):  
于潇宇 Yu Xiaoyu ◽  
郭玉波 Guo Yubo ◽  
陈刚 Chen Gang ◽  
叶东 Ye Dong
Keyword(s):  
Feature Extraction ◽  
Distributed Computing ◽  
Real Time ◽  
Connected Components ◽  
Point Feature ◽  
Time Point

scholarly journals Two Reliable Methodical Approaches for Non-Invasive RHD Genotyping of a Fetus from Maternal Plasma

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 564
Author(s):  
Jana Bohmova ◽  
Marek Lubusky ◽  
Iva Holuskova ◽  
Martina Studnickova ◽  
Romana Kratochvilova ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Real Time ◽  
Real Time Pcr ◽  
Dna Analysis ◽  
Methodological Approach ◽  
Maternal Plasma ◽  
Buccal Swab ◽  
Non Invasive ◽  
Pcr Method ◽  
Parallel Reactions

Noninvasive fetal RHD genotyping is an important tool for predicting RhD incompatibility between a pregnant woman and a fetus. This study aimed to assess a methodological approach other than the commonly used one for noninvasive fetal RHD genotyping on a representative set of RhD-negative pregnant women. The methodology must be accurate, reliable, and broadly available for implementation into routine clinical practice. A total of 337 RhD-negative pregnant women from the Czech Republic region were tested in this study. The fetal RHD genotype was assessed using two methods: real-time PCR and endpoint quantitative fluorescent (QF) PCR. We used exon-7-specific primers from the RHD gene, along with internal controls. Plasma samples were analyzed and measured in four/two parallel reactions to determine the accuracy of the RHD genotyping. The RHD genotype was verified using DNA analysis from a newborn buccal swab. Both methods showed an excellent ability to predict the RHD genotype. Real-time PCR achieved its greatest accuracy of 98.6% (97.1% sensitivity and 100% specificity (95% CI)) if all four PCRs were positive/negative. The QF PCR method also achieved its greatest accuracy of 99.4% (100% sensitivity and 98.6% specificity (95% CI)) if all the measurements were positive/negative. Both real-time PCR and QF PCR were reliable methods for precisely assessing the fetal RHD allele from the plasma of RhD-negative pregnant women.

scholarly journals Real-Time Sequencing of Mycobacterium tuberculosis: Are We There Yet?

2017 ◽  
Vol 55 (5) ◽  
pp. 1249-1254 ◽  
Author(s):  
Robyn S. Lee ◽  
Madhukar Pai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Real Time ◽  
Phylogenetic Trees ◽  
Illumina Miseq ◽  
Turnaround Time ◽  
Epidemiologic Studies ◽  
Content Type ◽  
Leading Role ◽  
Oxford Nanopore ◽  
Oxford Nanopore Technologies

ABSTRACT Whole-genome sequencing has taken a leading role in epidemiologic studies of tuberculosis, but thus far, its real-time clinical utility has been low, in part because of the requirement for culture. In their report in this issue, Votintseva et al. (A. A. Votintseva, P. Bradley, L. Pankhurst, C. del Ojo Elias, M. Loose, K. Nilgiriwala, A. Chatterjee, E. G. Smith, N. Sanderson, T. M. Walker, M. R. Morgan, D. H. Wyllie, A. S. Walker, T. E. A. Peto, D. W. Crook, and Z. Iqbal, J Clin Microbiol 55:1285–1298, 2017, https://doi.org/10.1128/JCM.02483-16 ) present a new method for extracting Mycobacterium tuberculosis DNA directly from smear-positive respiratory samples, making it feasible to generate drug resistance predictions and phylogenetic trees in 44 h with the Illumina MiSeq. They also illustrate the potential for a <24-h turnaround time from DNA extraction to clinically relevant results with Illumina MiniSeq and Oxford Nanopore Technologies MinION. We comment on the promise and limitations of these approaches.

Sign in / Sign up

Export Citation Format

Share Document