Evaluation of an effective detection and quantification method for particular microorganisms by comparing NGS-based metagenome profiling data

Abstract Metagenome profiling research using next-generation sequencing (NGS), a technique widely used to analyze the diversity and composition of microorganisms living in the human body, especially the gastrointestinal tract, has been activated, and there is a growing interest in the quantitative and diagnostic technology for specific microorganisms. According to recent trends, quantitative real-time PCR (qRT-PCR) are still of considerable technique in detecting and quantifying bacteria associated with the human mouth, nasal cavity, and pharynx due to analytical cost and time burden of NGS technology. Here, based on NGS metagenome profiling data produced by utilizing 100 gut microbiota samples, we conducted a comparative analysis of identifying for five bacterial genera proportions (Akkermansia, Bacteroides, Bifidobacterium, Phascolarctobacterium, and Roseburia) within same metagenomic DNA samples through qRT-PCR assay in parallel. Genus-specific primer, targeting the particular gene of each genus for qRT-PCR assay, allowed a statistically consistent quantification pattern with the metagenome profiling data. Furthermore, results of bacterial identification through Sanger validation demonstrated the high genus-specificity of each primer set. Therefore, our study suggests that an approach to quantifying specific microorganisms by applying qRT-PCR method can compensate for the concerns (potential issues) of NGS while also providing efficient benefits to various microbial industries

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Detection of Respiratory Pathogens in Parapneumonic Effusions by Hypothesis-free, Next-Generation Sequencing (NGS)

Open Forum Infectious Diseases ◽

10.1093/ofid/ofx162.044 ◽

2017 ◽

Vol 4 (suppl_1) ◽

pp. S17-S17

Author(s):

Krow Ampofo ◽

Andrew Pavia ◽

Anne J Blaschke ◽

Robert Schlaberg

Keyword(s):

Next Generation Sequencing ◽

Pathogen Detection ◽

Respiratory Pathogens ◽

Bacterial Identification ◽

Next Generation ◽

University Of Utah ◽

Species Specific ◽

Next Generation Sequencing Ngs ◽

Ngs Data ◽

Generation Sequencing

Abstract Background Species-specific polymerase chain reaction (PCR) testing of pleural fluid (PF) from children with parapneumonic effusion (PPE) has increased pathogen identification in pediatric PPE. However, a pathogen is not detected in 25–35% of cases. Hypothesis-free, next-generation sequencing (NGS) provides a more comprehensive alternative and has led to pathogen detection in PCR-negative samples. However, the utility of NGS in the evaluation of PF from children with PPE is unknown. Methods Archived PF (n = 20) from children younger than 18 years with PPE and hospitalized at Primary Children’s Hospital, Utah, in 2015 and previously tested by PCR were evaluated. Ten PCR-negative and 10 PCR-positive PF specimens were tested using RNA-seq at an average depth of 7.7×106 sequencing reads per sample. NGS data were analyzed with Taxonomer. We compared pathogens detected by blood and PF culture, PCR, and NGS. Results Overall, compared with blood/PF culture, PF PCR and PF NGS testing of PF increased bacterial identification from 15% to 50% (P < 0.05) and 65% (P = 0.003), respectively. Pathogen detection in PF by PCR and NGS were comparable (50 vs. 65%, p = NS) (Table). However, compared with PF PCR, NGS significantly increased detection of S. pyogenes (20% vs. 55%; P < 0.05), with 100% concordance when detected by PCR and culture. Detection of Fusobacterium spp. (10 vs. 10%) by PF NGS and PF PCR were comparable. In contrast, there was no detection of S. pneumoniae (15 vs. 0%) by PF NGS compared with PF PCR. Conclusion PF NGS testing significantly improves bacterial identification and comparable to PF PCR testing, which can help inform antimicrobial selection. However there were differences in detection of S. pneumoniae and S. pyogenes. Further studies of NGS testing of PF of children with PPE are needed to assess its potential in the evaluation of PPE in children. Disclosures A. J. Blaschke, BioFire Diagnostics LLC: Collaborator, Have intellectual property in BioFire Diagnostics through the University of Utah and Investigator, Licensing agreement or royalty and Research support; R. Schlaberg, IDbyDNA: Co-founder, Consultant and Shareholder, Stock

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Targeted Hybridization Capture of SARS-CoV-2 and Metagenomics Enables Genetic Variant Discovery and Nasal Microbiome Insights

10.1101/2021.03.16.21252988 ◽

2021 ◽

Author(s):

Dorottya Nagy-Szakal ◽

Mara Couto-Rodriguez ◽

Heather L. Wells ◽

Joseph E. Barrows ◽

Marilyne Debieu ◽

...

Keyword(s):

Genetic Variants ◽

Limit Of Detection ◽

Viral Evolution ◽

Viral Fitness ◽

Pcr Assay ◽

Hybridization Capture ◽

Variant Discovery ◽

Powerful Approach ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

The emergence of novel SARS-CoV-2 genetic variants that may alter viral fitness highlights the urgency of widespread next-generation sequencing (NGS) surveillance. To profile genetic variants, we developed and clinically validated a hybridization capture SARS-CoV-2 NGS assay, integrating novel methods for panel design using dsDNA biotin-labeled probes, and built accompanying software. The positive and negative percent agreement were defined in comparison to an orthogonal RT-PCR assay (PPA and NPA: both 96.7%). The limit of detection was established to be 800 copies/ml with an average fold-enrichment of 46,791x. We identified novel 107 mutations, including 24 in the functionally-important spike protein. Further, we profiled the full nasopharyngeal microbiome using metagenomics and found overrepresentation of 7 taxa and macrolide resistance in SARS-CoV-2-positive patients. This hybrid capture NGS assay, coupled with optimized software, is a powerful approach to detect and comprehensively map SARS-CoV-2 genetic variants for tracking viral evolution and guiding vaccine updates.

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Implementation of an in-house real-time reverse transcription-PCR assay for the rapid detection of the SARS-CoV-2 Marseille-4 variant

10.1101/2021.02.03.21250823 ◽

2021 ◽

Author(s):

Marielle Bedotto ◽

Pierre-Edouard Fournier ◽

Linda Houhamdi ◽

Anthony Levasseur ◽

Jeremy Delerce ◽

...

Keyword(s):

Immune Responses ◽

Real Time ◽

Reverse Transcription ◽

Geographical Area ◽

Viral Escape ◽

Pcr Assay ◽

Reverse Transcription Pcr ◽

One Step ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

ABSTRACTIntroductionThe SARS-CoV-2 pandemic has been associated with the occurrence since summer 2020 of several viral variants that overlapped or succeeded each other in time. Those of current concern harbor mutations within the spike receptor binding domain (RBD) that may be associated with viral escape to immune responses. In our geographical area a viral variant we named Marseille-4 harbors a S477N substitution in this RBD.Materials and methodsWe aimed to implement an in-house one-step real-time reverse transcription-PCR (qPCR) assay with a hydrolysis probe that specifically detects the SARS-CoV-2 Marseille-4 variant.ResultsAll 6 cDNA samples from Marseille-4 variant strains identified in our institute by genome next-generation sequencing (NGS) tested positive using our Marseille-4 specific qPCR, whereas all 32 cDNA samples from other variants tested negative. In addition, 39/42 (93%) respiratory samples identified by NGS as containing a Marseille-4 variant strain and 0/26 samples identified as containing non-Marseille-4 variant strains were positive. Finally, 1,585/2,889 patients SARS-CoV-2-diagnosed in our institute, 10/277 (3.6%) respiratory samples collected in Algeria, and none of 207 respiratory samples collected in Senegal, Morocco, or Lebanon tested positive using our Marseille-4 specific qPCR.DiscussionOur in-house qPCR system was found reliable to detect specifically the Marseille-4 variant and allowed estimating it is involved in more than half of our SARS-CoV-2 diagnoses since December 2020. Such approach allows the real-time surveillance of SARS-CoV-2 variants, which is warranted to monitor and assess their epidemiological and clinical characterics based on comprehensive sets of data.

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Clinical and Technical Aspects of Genomic Diagnostics for Precision Oncology

Journal of Clinical Oncology ◽

10.1200/jco.2016.70.7539 ◽

2017 ◽

Vol 35 (9) ◽

pp. 929-933 ◽

Cited By ~ 20

Author(s):

Yuri Sheikine ◽

Frank C. Kuo ◽

Neal I. Lindeman

Keyword(s):

Genomic Testing ◽

Precision Oncology ◽

Strategic Decisions ◽

Technical Aspects ◽

Testing Program ◽

Different Types ◽

Diagnostic Technology ◽

Next Generation Sequencing Ngs ◽

Build Systems ◽

Generation Sequencing

The emergence of precision medicine has been predicated on significant recent advances in diagnostic technology, particularly the advent of next-generation sequencing (NGS). Although the chemical technology underlying NGS is complex, and the computational biology expertise required to build systems to facilely interpret the results is highly specialized, the variables involved in designing and deploying a genomic testing program for cancer can be readily understood and applied by understanding several basic considerations. In this review, we present key strategic decisions required to optimize a genomic testing program and summarize the technical aspects of different technologies that render those methods more or less suitable for different types of programs.

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Utilization of a SARS-CoV-2 Variant Assay for the Rapid Differentiation of Omicron and Delta

10.1101/2021.12.22.21268195 ◽

2021 ◽

Author(s):

Nicholas Jacob Barasch ◽

James Iqbal ◽

Marvin Coombs ◽

Sofia Kazi ◽

Jessica Wang-Rodriguez ◽

...

Keyword(s):

Next Generation Sequencing ◽

Severe Acute Respiratory Syndrome ◽

Therapeutic Intervention ◽

Next Generation ◽

Rt Pcr ◽

Pcr Assay ◽

Spike Gene ◽

Next Generation Sequencing Ngs ◽

Generation Sequencing

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant (B.1.1.529), creates a diagnostic vacuum, since differentiation of Omicron from Delta relies on relatively slow next generation sequencing (NGS) technology delaying epidemiologic understanding and therapeutic intervention. The RUO SARS-CoV-2 Variant Set 1 Test (RSCov2V1) RT-PCR for detection of spike gene N501Y, E484K and del69-70 was designed to differentiate Alpha from Beta and Gamma variants. While Delta lacks these three variants, Omicron has the N501Y and del69-70 mutation. We submitted 88 samples for RSCov2V1 identifying 9 samples with the N501Y and del69-70 mutations while all other samples (79) were negative for all three variants. 9/9 samples with the del69-70 and N501Y were identified by NGS to be Omicron while 47/47 other samples assessed by NGS were confirmed to be Delta family variants. We demonstrate here that an immediately available RT-PCR assay for detection of spike gene N501Y and del69-70 can be utilized to rapidly differentiate Omicron from Delta variants in the proper epidemiologic context

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Upregulation of Circulating miR-195-3p in Heart Failure

Cardiology ◽

10.1159/000476029 ◽

2017 ◽

Vol 138 (2) ◽

pp. 107-114 ◽

Cited By ~ 13

Author(s):

Xia He ◽

Jun Ji ◽

Tao Wang ◽

Ming-Bang Wang ◽

Xiao-Ling Chen

Keyword(s):

Heart Failure ◽

Characteristic Curve ◽

Area Under The Curve ◽

Circulating Micrornas ◽

Qrt Pcr ◽

Potential Biomarker ◽

Patients With Heart Failure ◽

Pcr Method ◽

Next Generation Sequencing Ngs ◽

Plasma Rna

Objectives: Many circulating microRNAs (miRs) have been shown to have potential biomarker effects in cardiovascular disease. We studied the dysregulation of circulating miR-195-3p in patients with heart failure (HF) to elucidate its value as a potential biomarker for HF. Methods: Eight ischemic HF (IHF) patients, 8 nonischemic HF patients (NIHF), and 8 healthy volunteers (matched by age and sex - normal controls [NCs]) were chosen for miR sequencing. The plasma RNA was extracted, and a small RNA library of HF was established and then sequenced using next-generation sequencing (NGS) technology. The miR-195-3p was selected for a second clinical study in 60 IHF, 48 NIHF patients, and 35 NCs for qRT-PCR validation. Results: The expression of circulating miR-195-3p in the IHF group was increased 69.5-fold compared with the NC group using NGS technique, and it was the most elevated in all upregulated miRs. MiR-195-3p was ranked in the top 1 of all upregulated miRs in contribution to HF based on a random forest model analysis. The upregulation of circulating miR-195-3p was also validated with the qRT-PCR method, and receiver operating characteristic curve analysis showed that the area under the curve (AUC) was 0.831. Conclusions: The circulating miR-195-3p was upregulated in IHF and NIHF patients and could be a potential biomarker for HF.

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