Viral population analysis and minority-variant detection using short read next-generation sequencing

Watson, Simon J.; Welkers, Matthijs R.A.; Depledge, Daniel P.; Coulter, Eve; Breuer, Judith; Jong, Menno D. de; Kellam, Paul

doi:10.1098/rstb.2012.0205

Cited by 164 publications

(156 citation statements)

References 19 publications

Supporting

Mentioning

152

Contrasting

Order By: Relevance

“…Viral RNA was RT-PCR amplified using a universal 8-segment PCR method as described previously (25). In short, two separate RT-PCRs were performed for each sample, using primers common-uni12R (5=-GCCGGAGCTCTGCAGAT ATCAGCRAAAGCAGG-3=), common-uni12G (5=-GCCGGAGCTCTG CAGATATCAGCGAAAGCAGG-3=), and common-uni13 (5=-CAGGAA ACAGCTATGACAGTAGAAACAAGG-3=).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Emergence of the Virulence-Associated PB2 E627K Substitution in a Fatal Human Case of Highly Pathogenic Avian Influenza Virus A(H7N7) Infection as Determined by Illumina Ultra-Deep Sequencing

Jonges

Welkers

Jeeninga

et al. 2014

J Virol

Self Cite

View full text Add to dashboard Cite

dAvian influenza viruses are capable of crossing the species barrier and infecting humans. Although evidence of human-to-human transmission of avian influenza viruses to date is limited, evolution of variants toward more-efficient human-to-human transmission could result in a new influenza virus pandemic. In both the avian influenza A(H5N1) and the recently emerging avian influenza A(H7N9) viruses, the polymerase basic 2 protein (PB2) E627K mutation appears to be of key importance for human adaptation. During a large influenza A(H7N7) virus outbreak in the Netherlands in 2003, the A(H7N7) virus isolated from a fatal human case contained the PB2 E627K mutation as well as a hemagglutinin (HA) K416R mutation. In this study, we aimed to investigate whether these mutations occurred in the avian or the human host by Illumina Ultra-Deep sequencing of three previously uninvestigated clinical samples obtained from the fatal case. In addition, we investigated three chicken samples, two of which were obtained from the source farm. Results showed that the PB2 E627K mutation was not present in any of the chicken samples tested. Surprisingly, the avian samples were characterized by the presence of influenza virus defective RNA segments, suggestive for the synthesis of defective interfering viruses during infection in poultry. In the human samples, the PB2 E627K mutation was identified with increasing frequency during infection. Our results strongly suggest that human adaptation marker PB2 E627K has emerged during virus infection of a single human host, emphasizing the importance of reducing human exposure to avian influenza viruses to reduce the likelihood of viral adaptation to humans.

show abstract

Section: Methodsmentioning

confidence: 99%

“…(i) Demultiplexing. The raw fastq files containing the sequences with corresponding quality scores were split into separate sample-specific files based on the corresponding MID sequence using the readset parser function of the Quality Assessment of Short Reads (QUASR) package version 7.0.1 (25).…”

Section: Illumina Sequencing (I) Genome Analyzer Iix (Gaiix)mentioning

confidence: 99%

Emergence of the Virulence-Associated PB2 E627K Substitution in a Fatal Human Case of Highly Pathogenic Avian Influenza Virus A(H7N7) Infection as Determined by Illumina Ultra-Deep Sequencing

Jonges

Welkers

Jeeninga

et al. 2014

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Following demultiplexing, each sequence dataset was profiled using FastQC (www.bioinformatics.babraham. ac.uk/projects/fastqc/) and parsed through QUASR (34) and Trim Galore for duplicate removal and read-trimming, respectively. Sequence reads were aligned against the VZV reference strain POka (AB097933) using BurrowsWheeler alignment (35).…”

Section: Significancementioning

confidence: 99%

In vitro system using human neurons demonstrates that varicella-zoster vaccine virus is impaired for reactivation, but not latency

Sadaoka

Depledge

Rajbhandari

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

104

View full text Add to dashboard Cite

Varicella-zoster virus (VZV) establishes latency in human sensory and cranial nerve ganglia during primary infection (varicella), and the virus can reactivate and cause zoster after primary infection. The mechanism of how the virus establishes and maintains latency and how it reactivates is poorly understood, largely due to the lack of robust models. We found that axonal infection of neurons derived from hESCs in a microfluidic device with cell-free parental Oka (POka) VZV resulted in latent infection with inability to detect several viral mRNAs by reverse transcriptase-quantitative PCR, no production of infectious virus, and maintenance of the viral DNA genome in endless configuration, consistent with an episome configuration. With deep sequencing, however, multiple viral mRNAs were detected. Treatment of the latently infected neurons with Ab to NGF resulted in production of infectious virus in about 25% of the latently infected cultures. Axonal infection of neurons with vaccine Oka (VOka) VZV resulted in a latent infection similar to infection with POka; however, in contrast to POka, VOka-infected neurons were markedly impaired for reactivation after treatment with Ab to NGF. In addition, viral transcription was markedly reduced in neurons latently infected with VOka compared with POka. Our in vitro system recapitulates both VZV latency and reactivation in vivo and may be used to study viral vaccines for their ability to establish latency and reactivate. varicella-zoster virus | varicella vaccine | reactivation | latency | herpesvirus V aricella-zoster virus (VZV) is a member of Alphaherpesvirinae, characterized by neurotropism and lifelong latent infection in human dorsal root, cranial nerve, and enteric ganglia (1). During primary infection (varicella), VZV gains access to sensory neurons by two potential routes: retrograde axonal transport from cutaneous lesions or infection of neurons by virus-infected T cells circulating through the body (2). The virus then establishes latency in neurons, and months to years later, when VZV-specific T cells decline, the virus can reactivate to cause herpes zoster.VZV is the only human herpesvirus for which a licensed vaccine is approved, and the live-attenuated vaccine Oka (VOka) virus is effective in preventing both varicella and zoster. VOka was derived from WT parental Oka (POka) by propagation in human embryonic lung cells, guinea pig embryo fibroblasts, and human fibroblasts (3, 4). VOka is a genetic mixture of at least eight genotypes (5), and, initially, the genomes of POka and VOka were found to differ by 42 nucleotides and 20 amino acids (6). A recent study using deep sequencing identified 165 additional nucleotide changes in VOka, although most changes were presence in <10% of viral genomes (7). Attenuation of VOka is postulated to be due to mutations in multiple regions of the genome (8, 9).Despite numerous studies, the mechanisms for establishment and maintenance of VZV latency and subsequent reactivation remain poorly understood, primarily due to the lack of ...

show abstract

“…As the activity of the transposases generally results in nonrandom integrations (22,23), some biases in sequence coverage may arise if the distribution of the fragmentation and tagging is not homogeneous throughout the genome or between genotypes. Finally, sequencing errors occur whatever the method used, and appropriate data analyses are required to distinguish them from actual polymorphisms (24)(25)(26).…”

Section: Importancementioning

confidence: 99%

The Number of Target Molecules of the Amplification Step Limits Accuracy and Sensitivity in Ultradeep-Sequencing Viral Population Studies

Gallet

Fabre

Michalakis

et al. 2017

J Virol

View full text Add to dashboard Cite

The invention of next-generation sequencing (NGS) techniques marked the coming of a new era in the detection of the genetic diversity of intrahost viral populations. A good understanding of the genetic structure of these populations requires, first, the ability to identify the different isolates or variants and, second, the ability to accurately quantify them. However, the initial amplification step of NGS studies can impose potential quantitative biases, modifying the variant relative frequencies. In particular, the number of target molecules (NTM) used during the amplification step is vastly overlooked although of primary importance, as it sets the limit of the accuracy and sensitivity of the sequencing procedure. In the present article, we investigated quantitative biases in an NGS study of populations of a multipartite single-stranded DNA (ssDNA) virus at different steps of the procedure. We studied 20 independent populations of the ssDNA virus faba bean necrotic stunt virus (FBNSV) in two host plants, Vicia faba and Medicago truncatula. FBNSV is a multipartite virus composed of eight genomic segments, whose specific and hostdependent relative frequencies are defined as the "genome formula." Our results show a significant distortion of the FBNSV genome formula after the amplification and sequencing steps. We also quantified the genetic bottleneck occurring at the amplification step by documenting the NTM of two genomic segments of FBNSV. We argue that the NTM must be documented and carefully considered when determining the sensitivity and accuracy of data from NGS studies.IMPORTANCE The advent of next-generation sequencing (NGS) techniques now enables study of the genetic diversity of viral populations. A good understanding of the genetic structure of these populations first requires the ability to identify the different isolates or variants and second requires the ability to accurately quantify them. Prior to sequencing, viral genomes need to be amplified, a step that potentially imposes quantitative biases and modifies the viral population structure. In particular, the number of target molecules (NTM) used during the amplification step is of primary importance, as it sets the limit of the accuracy and sensitivity of the sequencing procedure. In this work, we used 20 replicated populations of the multipartite faba bean necrotic stunt virus (FBNSV) to estimate the various limitations of ultradeep-sequencing studies performed on intrahost viral populations. We report quantitative biases during rolling-circle amplification and the NTM of two genomic segments of FBNSV.KEYWORDS DNA sequencing, FBNSV, faba bean, Medicago truncatula, nextgeneration sequencing, number of target molecules, rolling-circle amplification, sequencing accuracy, sequencing sensitivity

show abstract

Viral population analysis and minority-variant detection using short read next-generation sequencing

Cited by 164 publications

References 19 publications

Emergence of the Virulence-Associated PB2 E627K Substitution in a Fatal Human Case of Highly Pathogenic Avian Influenza Virus A(H7N7) Infection as Determined by Illumina Ultra-Deep Sequencing

Emergence of the Virulence-Associated PB2 E627K Substitution in a Fatal Human Case of Highly Pathogenic Avian Influenza Virus A(H7N7) Infection as Determined by Illumina Ultra-Deep Sequencing

In vitro system using human neurons demonstrates that varicella-zoster vaccine virus is impaired for reactivation, but not latency

The Number of Target Molecules of the Amplification Step Limits Accuracy and Sensitivity in Ultradeep-Sequencing Viral Population Studies

Contact Info

Product

Resources

About