Insights from Genomics into Bacterial Pathogen Populations

doi:10.1201/b17137-19

Cited by 40 publications

(54 citation statements)

References 31 publications

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“…Furthermore, It has been shown that mutation errors in pyrosequencing reads tend to occur predominantly in homopolymeric regions (17,27). In the present study we did not assess the error rate of the present assay; however, others have estimated the overall error rate of the pyrosequencing technique to be on the average of 0.3% (12,28). It was also previously shown that the pyrosequencing error rate for detection of 17 known drug resistance mutations, located at homopolymer regions in the PR and RT genes of subtype B viruses, was Ͻ0.71% (17).…”

Section: Discussionmentioning

confidence: 42%

“…A later platform, the gene sequencer (GS) FLX pyrosequencing system is capable of producing 400 to 600 million bases per 10-h run (11). This technology also has a relatively long sequencing read length of 400 to 600 bp, which has the added advantage over the point mutation assays of being able to detect mutations in their sequence context and not just at a single locus (12).…”

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confidence: 99%

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Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory

et al. 2013

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f Detection of low-abundance drug resistance mutations (DRMs) of HIV-1 is an evolving approach in clinical practice. Ultradeep pyrosequencing has shown to be effective in detecting such mutations. The lack of a standardized commercially based assay limits the wide use of this method in clinical settings. 454 Life Sciences (Roche) is developing an HIV ultradeep pyrosequencing assay for their benchtop sequencer. We assessed the prototype plate in the clinical laboratory. Plasma samples genotyped by the standardized TruGene kit were retrospectively tested by this assay. Drug-treated subjects failing therapy and drug-naive patients were included. DRM analysis was based on the International AIDS Society USA DRM list and the Stanford algorithm. The prototype assay detected all of the DRMs detected by TruGene and additional 50 low-abundance DRMs. Several patients had lowabundance D67N, K70R, and M184V reverse transcriptase inhibitor mutations that persisted long after discontinuation of the drug that elicited these mutations. Additional patient harbored low-abundance V32I major protease inhibitor mutation, which under darunavir selection evolved later to be detected by TruGene. Stanford analysis suggested that some of the low-abundance DRMs were likely to affect the resistance burden in these subjects. The prototype assay performs at least as well as TruGene and has the advantage of detecting low-abundance drug resistance mutations undetected by TruGene. Its ease of use and lab-scale platform will likely facilitate its use in the clinical laboratory. The extent to which the detection of low-abundance DRMs will affect patient management is still unknown, but it is hoped that use of such an assay in clinical practice will help resolve this important question.

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Section: Discussionmentioning

confidence: 42%

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confidence: 99%

Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory

et al. 2013

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“…Furthermore, the available studies on WNV mutant spectrum diversification relied on Sanger sequencing methods (Deardorff et al, 2011;Jerzak et al, 2005Jerzak et al, , 2007Jerzak et al, , 2008, whereby a limited set of consensus sequences was obtained after a plaque-purification or molecular cloning step. Nextgeneration sequencing (NGS) technologies present an opportunity to deepen the analysis of RNA virus genetic variants (Barzon et al, 2011;Van Borm et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Next-generation sequencing shows West Nile virus quasispecies diversification after a single passage in a carrion crow (Corvus corone) in vivo infection model

Dridi

Rosseel

Orton

et al. 2015

Journal of General Virology

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West Nile virus (WNV) occurs as a population of genetic variants (quasispecies) infecting a single animal. Previous low-resolution viral genetic diversity estimates in sampled wild birds and mosquitoes, and in multiple-passage adaptation studies in vivo or in cell culture, suggest that WNV genetic diversification is mostly limited to the mosquito vector. This study investigated genetic diversification of WNV in avian hosts during a single passage using next-generation sequencing. Wild-captured carrion crows were subcutaneously infected using a clonal MiddleEast WNV. Blood samples were collected 2 and 4 days post-infection. A reverse-transcription (RT)-PCR approach was used to amplify the WNV genome directly from serum samples prior to next-generation sequencing resulting in an average depth of at least 7006 in each sample. Appropriate controls were sequenced to discriminate biologically relevant low-frequency variants from experimentally introduced errors. The WNV populations in the wild crows showed significant diversification away from the inoculum virus quasispecies structure. By contrast, WNV populations in intracerebrally infected day-old chickens did not diversify from that of the inoculum. Where previous studies concluded that WNV genetic diversification is only experimentally demonstrated in its permissive insect vector species, we have experimentally shown significant diversification of WNV populations in a wild bird reservoir species.

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“…[13][14][15] This approach is cost effective and highly efficient for the recovery of viral genome sequences, taking advantage of the detection and characterization of viral genomes and viral metagenomics. 16,17 Sequence-independent, single-primer amplification (SISPA) is a methodology to synthesize and enrich viral genomes using random hexamer and SISPA sequences (5′-GCCGGAGCTCTGCAGATATC-3′). 18 SISPA NGS was used for defining RNA and DNA viruses including Rotavirus, Astrovirus, and Parvovirus.…”

Section: Introductionmentioning

confidence: 99%

Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture–Based Isolates

Song

Kim

et al. 2017

The American Society of Tropical Medicine and Hygiene

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Abstract. Hantaan virus (HTNV), identified in the striped field mouse (Apodemus agrarius), belongs to the genus Hantavirus of the family Bunyaviridae and contains tripartite RNA genomes, small (S), medium (M), and large (L) segments. HTNV is a major causative for hemorrhagic fever with renal syndrome (HFRS) with fatality rates ranging from 1% to 15% in the Republic of Korea (ROK) and China. Defining of HTNV whole-genome sequences and isolation of the infectious particle play a critical role in the characterization and preventive and therapeutic strategies of hantavirus outbreaks. Next-generation sequencing (NGS) provides an advanced tool for massive genomic sequencing of viruses. However, the isolation of viral infectious particles is a huge obstacle to investigate and develop anti-virals for hantaviruses. Here, we report 12 HTNV isolates from lung tissues of the striped field mouse in the highly HFRS-endemic areas. Sequence-independent, single-primer amplification (SISPA) NGS was attempted to recover the genomic sequences of HTNV isolates. The nucleotide sequence of HTNV S, M, and L segments were covered up to 99.4-100%, 97.5-100%, and 95.6-99.8%, respectively, based on the full length of the prototype HTNV 76-118. The whole-genome sequencing of HTNV isolates was accomplished by additional reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification cDNA ends (RACE) PCR. In conclusion, this study will lead to the attempt and usage of SISPA NGS technologies to delineate the whole-genome sequence of hantaviruses, providing a new era of viral genomics for the surveillance, trace, and disease risk management of HFRS incidents.

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Insights from Genomics into Bacterial Pathogen Populations

Cited by 40 publications

References 31 publications

Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory

Evaluation of a Benchtop HIV Ultradeep Pyrosequencing Drug Resistance Assay in the Clinical Laboratory

Next-generation sequencing shows West Nile virus quasispecies diversification after a single passage in a carrion crow (Corvus corone) in vivo infection model

Sequence-Independent, Single-Primer Amplification Next-Generation Sequencing of Hantaan Virus Cell Culture–Based Isolates

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