Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

Greninger, Alexander L.; Naccache, Samia N.; Federman, Scot; Yu, Guixia; Mbala, Placide; Brès, Vanessa; Stryke, Doug; Bouquet, Jérôme; Somasekar, Sneha; Linnen, Jeffrey M.; Dodd, Roger Y.; Mulembakani, Prime; Schneider, Bradley S.; Muyembe‐Tamfum, Jean‐Jacques; Stramer, Susan L.; Chiu, Charles Y.

doi:10.1186/s13073-015-0220-9

Cited by 480 publications

(391 citation statements)

References 38 publications

(58 reference statements)

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“…Traditionally, barriers to NGS implementation have included high costs, complex instrumentation, and lack of dedicated bioinformatic tools. These barriers are being overcome with the development of rapid computational pipelines for analysis of mNGS data (9,22,23), as well as emergence of portable sequencers that can be used in field laboratories and other point-of-care settings (24,25). The establishment of robust cutoff thresholds for determining positive results will also be needed before mNGS can be used routinely for infectious disease diagnosis.…”

Section: Discussionmentioning

confidence: 99%

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

et al. 2016

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Zika virus (ZIKV), a flavivirus, and chikungunya virus (CHIKV), an alphavirus, are infectious RNA arboviruses transmitted to humans by the bite of Aedes species mosquitoes. Both viruses have only recently emerged in the Western Hemisphere (1, 2), and along with dengue virus (DENV), another flavivirus, now circulate widely in Brazil. The acute illness caused by these viruses, characterized by fever, rash, myalgia, arthralgia, and conjunctivitis, is nonspecific, and differential diagnosis on the basis of clinical findings alone is challenging. Later infectious sequelae include chronic arthritis for CHIKV (2) and encephalitis, immune-mediated syndromes, and stroke for DENV (3). Recently, the association between ZIKV infection and severe fetal complications such as microcephaly in pregnant women has been established (4), and the virus has also been linked to neurological complications such as Guillain-Barré syndrome (5). Thus, broadbased assays are needed for differential diagnosis of vectorborne febrile illnesses and to identify potential coinfections. Here we report the utility of metagenomic next-generation sequencing (mNGS) as a screening tool to identify coinfections and the use of genome recovery and phylogenetic analyses directly from patient serum samples in the context of the ongoing ZIKV outbreak. We also show that the clinical presentation of arboviral coinfections seems to favor the virus present at a higher titer in acutely infected individuals. MATERIALS AND METHODSZIKV serum sample collection, ZIKV RT-PCR, and DENV antibody testing. Written consent from patients was obtained under a study protocol approved by the Brazil Ministry of Health (Certificado de Apresentação para Apreciação Ética 45483115.0.0000.0046, no. 1159.184, Brazil). Serum samples were obtained from 15 patients seen at Aliança Hospital in Salvador, Bahia, Brazil, from April 2015 to January 2016 who were given a presumptive diagnosis of an acute viral illness by emergency department physicians and were found to be positive by qualitative reverse transcription-PCR (RT-PCR) testing for ZIKV. Serum samples Bahia01 to Bahia15 were subjected to RNA extraction using the QIAamp viral RNA minikit (Qiagen), and RNA was reverse transcribed using the Superscript II reverse transcriptase kit (Invitrogen), followed by qualitative RT-PCR testing for ZIKV using primers targeting the NS5 gene (6). Serum samples were also tested for DENV infection using an enzyme-linked immunosorbent assay (ELISA) specific for the NS1 antigen and anti-DENV IgG/IgM according to the manufacturer's instructions (Dengue Duo Test; Bioeasy Diagnostica, Brazil).Metagenomic next-generation sequencing. A separate serum aliquot was extracted for total nucleic acid using the Qiagen viral RNA minikit (Qiagen), followed by DNase treatment using a cocktail of Turbo DNase (Thermo Fisher Scientific) and Baseline-Zero DNase (Epicentre Biotechnologies), followed by NGS library construction using the NexteraXT kit (Illumina) as previously described (7,8). Runs of single-end, 160-base pair...

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Section: Discussionmentioning

confidence: 99%

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

et al. 2016

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“…Despite the relatively high error rate, MinION reads are suitable for de novo assembly of complete genomes (15,16,17,18), scaffolding NGS contigs (19), metagenomic studies (20,21), and realtime epidemiological investigations (22,23). The main advantages of the MinION platform over NGS technologies are the long reads (there is no theoretical limit of read length), the low investment cost per device, portability, and the flexible run and reduced turnaround time (22,24). Plasmids R16a and IP40a (R40a) (25) were isolated in the Pasteur Institute from abscess and urine samples collected in 1966 (St-Antoine Hospital, Paris, France) and 1969 (Necker Hospital, Paris, France), respectively.…”

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confidence: 99%

Characterization of Two Multidrug-Resistant IncA/C Plasmids from the 1960s by Using the MinION Sequencer Device

Szabó

Nagy

Wilk

et al. 2016

Antimicrob Agents Chemother

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Two A/C incompatibility group (IncA/C family) plasmids from the 1960s have been sequenced and classified into the A/C 2 type 1 group. R16a and IP40a contain novel antibiotic resistance islands and a complete GIsul2 genomic island not previously found in the family. In the 173.1-kb R16a, the 29.9-kb antibiotic resistance island (ARI) is located in a unique backbone position not utilized by ARIs. ARI R16a consists of Tn1, Tn6020, and Tn6333, harboring the resistance genes bla TEM-1D and aphA1b and a mer module, respectively; a truncated Tn5393 copy; and a gene cluster with unknown function. Plasmid IP40a is 170.4 kb in size and contains a 5.6-kb ARI inserted into the kfrA gene. ARI IP40a carrying bla TEM-1D and aphA1b genes is composed of Tn1 with a Tn6023 insertion. Additionally, IP40a harbors single IS2, IS186, and Tn1000 insertions scattered in the backbone; an IS150 copy in GIsul2; and a complete Tn6333 carrying a mer module at the position of ARI R16a . Loss of resistance markers in R16a, IP40a, and R55 was observed during stability tests. Every phenotypic change proved to be the result of recombination events involving mobile elements. Intramolecular transposition of IS copies that generated IP40a derivatives lacking large parts of the backbone could account for the formation of other family members, too. The MinION platform proved to be a valuable tool in bacterial genome sequencing since it generates long reads that span repetitive elements and facilitates full-length plasmid or chromosome assembly. Nanopore technology enables rapid characterization of large, low-copy-number plasmids and their rearrangement products.

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“…The AD-PCR-measured mutation percentages for the three samples were 28%, 33%, and 48%, respectively (Figure 5c), consistent with the nanopore result. 27 …”

Section: Resultsmentioning

confidence: 99%

Nanolock–Nanopore Facilitated Digital Diagnostics of Cancer Driver Mutation in Tumor Tissue

et al. 2017

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Cancer driver mutations are clinically significant biomarkers. In precision medicine, accurate detection of these oncogenic changes in patients would enable early diagnostics of cancer, individually tailored targeted therapy, and precise monitoring of treatment response. Here we investigated a novel nanolock−nanopore method for single-molecule detection of a serine/threonine protein kinase gene BRAF V600E mutation in tumor tissues of thyroid cancer patients. The method lies in a noncovalent, mutation sequence-specific nanolock. We found that the nanolock formed on the mutant allele/probe duplex can separate the duplex dehybridization procedure into two sequential steps in the nanopore. Remarkably, this stepwise unzipping kinetics can produce a unique nanopore electric marker, with which a single DNA molecule of the cancer mutant allele can be unmistakably identified in various backgrounds of the normal wild-type allele. The single-molecule sensitivity for mutant allele enables both binary diagnostics and quantitative analysis of mutation occurrence. In the current configuration, the method can detect the BRAF V600E mutant DNA lower than 1% in the tumor tissues. The nanolock−nanopore method can be adapted to detect a broad spectrum of both transversion and transition DNA mutations, with applications from diagnostics to targeted therapy.

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Rapid metagenomic identification of viral pathogens in clinical samples by real-time nanopore sequencing analysis

Cited by 480 publications

References 38 publications

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

Coinfections of Zika and Chikungunya Viruses in Bahia, Brazil, Identified by Metagenomic Next-Generation Sequencing

Characterization of Two Multidrug-Resistant IncA/C Plasmids from the 1960s by Using the MinION Sequencer Device

Nanolock–Nanopore Facilitated Digital Diagnostics of Cancer Driver Mutation in Tumor Tissue

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