Deep sequencing: Becoming a critical tool in clinical virology

Quiñones‐Mateu, Miguel E.; Ávila, S.; Reyes‐Terán, Gustavo; Martı́nez, Miguel Ángel

doi:10.1016/j.jcv.2014.06.013

Cited by 115 publications

(87 citation statements)

References 232 publications

(186 reference statements)

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“…Despite this depth being in the lower range of those of most NGS technologies, it was sufficient to capture the within-host dynamics of viral genetic diversity and divergence in great detail. Moreover, with this number of full passes, the error rate was 0.37%, which is well below the general accepted error rate of 1% for NGS technologies (19).…”

Section: Discussionmentioning

confidence: 50%

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing

Raghwani

Koekkoek

et al. 2017

J Virol

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In contrast to other available next-generation sequencing platforms, PacBio single-molecule, real-time (SMRT) sequencing has the advantage of generating long reads albeit with a relatively higher error rate in unprocessed data. Using this platform, we longitudinally sampled and sequenced the hepatitis C virus (HCV) envelope genome region (1,680 nucleotides [nt]) from individuals belonging to a cluster of sexually transmitted cases. All five subjects were coinfected with HIV-1 and a closely related strain of HCV genotype 4d. In total, 50 samples were analyzed by using SMRT sequencing. By using 7 passes of circular consensus sequencing, the error rate was reduced to 0.37%, and the median number of sequences was 612 per sample. A further reduction of insertions was achieved by alignment against a sample-specific reference sequence. However, in vitro recombination during PCR amplification could not be excluded. Phylogenetic analysis supported close relationships among HCV sequences from the four male subjects and subsequent transmission from one subject to his female partner. Transmission was characterized by a strong genetic bottleneck. Viral genetic diversity was low during acute infection and increased upon progression to chronicity but subsequently fluctuated during chronic infection, caused by the alternate detection of distinct coexisting lineages. SMRT sequencing combines long reads with sufficient depth for many phylogenetic analyses and can therefore provide insights into within-host HCV evolutionary dynamics without the need for haplotype reconstruction using statistical algorithms.IMPORTANCE Next-generation sequencing has revolutionized the study of genetically variable RNA virus populations, but for phylogenetic and evolutionary analyses, longer sequences than those generated by most available platforms, while minimizing the intrinsic error rate, are desired. Here, we demonstrate for the first time that PacBio SMRT sequencing technology can be used to generate full-length HCV envelope sequences at the single-molecule level, providing a data set with large sequencing depth for the characterization of intrahost viral dynamics. The selection of consensus reads derived from at least 7 full circular consensus sequencing rounds significantly reduced the intrinsic high error rate of this method. We used this method to genetically characterize a unique transmission cluster of sexually transmitted HCV infections, providing insight into the distinct evolutionary pathways in each patient over time and identifying the transmission-associated genetic bottle-

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Section: Discussionmentioning

confidence: 50%

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing

Raghwani

Koekkoek

et al. 2017

J Virol

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“…27 Recently, the usage of NGS has tremendously increased in defining viral genomic sequences, outbreaks, and effective therapeutics. 10 The viral genomic recovery of hantavirus-infected hosts or patients is critical for the characterization of an endemic outbreak and disease risk mitigation, given that hantavirus infections cause serious diseases, such as HFRS and HPS. This report presents the implementation of SISPA NGS for sequencing and the genomic characterization HTNV isolates from highly HFRS-endemic areas.…”

Section: Resultsmentioning

confidence: 99%

“…[7][8][9][10] Usage of molecular genomic amplification, for example, polymerase chain reaction (PCR), is a representative method to acquire viral genome sequences from specimens. However, it is time and effort consuming to obtain the complete genome sequences of ultra-low copy of viral RNA in tissues or clinical samples.…”

Section: Introductionmentioning

confidence: 99%

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

Song

Kim

et al. 2016

Am J Trop Med Hyg

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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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“…Another study required 2 to 6 cDNA reactions and 5 to 35 separate PCRs to generate libraries for full-genome sequencing on three different NGS platforms (13). The speed of Nextera XT, the tremendous sequence depth, an error rate of 0.1%, and the current read lengths (v3 ϭ 600 nt) obtained on the ubiquitous Illumina MiSeq are also preferable to the time-consuming library amplification steps, lower throughput, and higher error rates associated with other platforms (11,36). We estimate the cost of the m2000 extraction, HIV-SMART library prep, and MiSeq sequencing reagent was approximately $165 per genome for the multiplex run of 23 libraries.…”

Section: Discussionmentioning

confidence: 99%

A Pan-HIV Strategy for Complete Genome Sequencing

et al. 2016

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bMolecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5= end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ϳ2,000؋ depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of <4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance.

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Deep sequencing: Becoming a critical tool in clinical virology

Cited by 115 publications

References 232 publications

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing

Characterization of Hepatitis C Virus (HCV) Envelope Diversification from Acute to Chronic Infection within a Sexually Transmitted HCV Cluster by Using Single-Molecule, Real-Time Sequencing

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

A Pan-HIV Strategy for Complete Genome Sequencing

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