Enhanced virome sequencing using targeted sequence capture

Wylie, Todd; Wylie, Kristine M.; Herter, Brandi; Storch, Gregory A.

doi:10.1101/gr.191049.115

Cited by 211 publications

(242 citation statements)

References 52 publications

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“…Specifically, mNGS was likely more sensitive for detection of CHIKV than the CHIKV PCR used in the current study, given that 8.6% and 9.8% of the viral genome was recovered by mNGS from two CHIKV-negative samples (negative by PCR), while mNGS was less sensitive or as sensitive as the ZIKV PCR assays (Table 1). Such discrepancies between mNGS and PCR at very low viral titers have been previously reported in the other metagenomic studies (26,27) and can potentially be addressed by formal clinical validation of mNGS assay performance and the use of rigorous negative and positive controls (28).…”

Section: Discussionmentioning

confidence: 93%

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: 93%

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

et al. 2016

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“…The nature of the sequencing protocol implies limited amplification of the viral genetic material, and a significant competition from the larger human genome. Therefore, this approach may not identify lower concentration viruses that could be revealed by using viral particles enrichment [58, 59] or viral genome capture [60, 61]. The latter methods rest on the ability to capture closely related sequences by hybridization to short conserved probes.…”

Section: Discussionmentioning

confidence: 99%

The blood DNA virome in 8,000 humans

Moustafa¹,

Xie²,

Kirkness³

et al. 2017

PLoS Pathog

266

258

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The characterization of the blood virome is important for the safety of blood-derived transfusion products, and for the identification of emerging pathogens. We explored non-human sequence data from whole-genome sequencing of blood from 8,240 individuals, none of whom were ascertained for any infectious disease. Viral sequences were extracted from the pool of sequence reads that did not map to the human reference genome. Analyses sifted through close to 1 Petabyte of sequence data and performed 0.5 trillion similarity searches. With a lower bound for identification of 2 viral genomes/100,000 cells, we mapped sequences to 94 different viruses, including sequences from 19 human DNA viruses, proviruses and RNA viruses (herpesviruses, anelloviruses, papillomaviruses, three polyomaviruses, adenovirus, HIV, HTLV, hepatitis B, hepatitis C, parvovirus B19, and influenza virus) in 42% of the study participants. Of possible relevance to transfusion medicine, we identified Merkel cell polyomavirus in 49 individuals, papillomavirus in blood of 13 individuals, parvovirus B19 in 6 individuals, and the presence of herpesvirus 8 in 3 individuals. The presence of DNA sequences from two RNA viruses was unexpected: Hepatitis C virus is revealing of an integration event, while the influenza virus sequence resulted from immunization with a DNA vaccine. Age, sex and ancestry contributed significantly to the prevalence of infection. The remaining 75 viruses mostly reflect extensive contamination of commercial reagents and from the environment. These technical problems represent a major challenge for the identification of novel human pathogens. Increasing availability of human whole-genome sequences will contribute substantial amounts of data on the composition of the normal and pathogenic human blood virome. Distinguishing contaminants from real human viruses is challenging.

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“…Although we only used representative genomes to generate our bait set, it is now well established that hybridization capture does not require perfect sequence match to function efficiently; for example, viral sequences exhibiting more than 40% divergence to the according baits have already been enriched (Wylie et al 2015). …”

Section: Methodsmentioning

confidence: 99%

Ancient Recombination Events between Human Herpes Simplex Viruses

Burrel

Boutolleau

Ryu

et al. 2017

Molecular Biology and Evolution

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Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) are seen as close relatives but also unambiguously considered as evolutionary independent units. Here, we sequenced the genomes of 18 HSV-2 isolates characterized by divergent UL30 gene sequences to further elucidate the evolutionary history of this virus. Surprisingly, genome-wide recombination analyses showed that all HSV-2 genomes sequenced to date contain HSV-1 fragments. Using phylogenomic analyses, we could also show that two main HSV-2 lineages exist. One lineage is mostly restricted to subSaharan Africa whereas the other has reached a global distribution. Interestingly, only the worldwide lineage is characterized by ancient recombination events with HSV-1. Our findings highlight the complexity of HSV-2 evolution, a virus of putative zoonotic origin which later recombined with its human-adapted relative. They also suggest that coinfections with HSV-1 and 2 may have genomic and potentially functional consequences and should therefore be monitored more closely.

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Enhanced virome sequencing using targeted sequence capture

Cited by 211 publications

References 52 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

The blood DNA virome in 8,000 humans

Ancient Recombination Events between Human Herpes Simplex Viruses

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