Hybrid Capture-Based Next Generation Sequencing and Its Application to Human Infectious Diseases

Gaudin, Maxime; Desnues, Christelle

doi:10.3389/fmicb.2018.02924

Cited by 93 publications

(66 citation statements)

References 82 publications

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“…Based on the data obtained in this study, we recommend multiplexing 48 samples per sequencing run. The use of targeted NGS for virus discovery is not new (15)(16)(17)19). Notably, Virocap and VirCapSeq-VERT are two well-established platforms targeting viruses that infect vertebrate hosts (15,17).…”

Section: Discussionmentioning

confidence: 99%

“…While the CoV enrichment NGS successfully identified nine new CoVs, the CoVspecific enrichment also has limitations. While other enrichment NGS approaches aim to identify a broad range of known viruses across the virome (15)(16)(17), our pipeline was designed to identify known and diverse CoVs. The most challenging region to sequence was the spike gene, which has the lowest bait coverage across the genome.…”

Section: Discussionmentioning

confidence: 99%

“…The genome references used in bait design do not fully reflect the diversity in this region. This is not unexpected, as this technology was not designed to detect completely novel viruses (15)(16)(17). One solution is to constantly update the baits in the CoV library to include sequence variations as they are reported (20).…”

Section: Discussionmentioning

confidence: 99%

“…Strategies to improve the efficiency of NGS have been explored, including subtraction of host genetic material or enrichment of viral nucleic acid through positive selection using a capture-based system, where the latter was proven more costeffective (15)(16)(17). Virus enrichment NGS has been successfully used for various viral families, and the most common protocols rely on predesigned viral probes that share more than 60% homology with the target virus sequence (15)(16)(17). In this study, we utilized an enrichment NGS approach with predesigned probes targeting most of the CoV species (18).…”

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confidence: 99%

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Discovery of Bat Coronaviruses through Surveillance and Probe Capture-Based Next-Generation Sequencing

Hao

Zhu

et al. 2020

mSphere

105

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Coronaviruses (CoVs) of bat origin have caused two pandemics in this century. Severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV both originated from bats, and it is highly likely that bat coronaviruses will cause future outbreaks. Active surveillance is both urgent and essential to predict and mitigate the emergence of these viruses in humans. Next-generation sequencing (NGS) is currently the preferred methodology for virus discovery to ensure unbiased sequencing of bat CoVs, considering their high genetic diversity. However, unbiased NGS is an expensive methodology and is prone to missing low-abundance CoV sequences due to the high background level of nonviral sequences present in surveillance field samples. Here, we employ a capture-based NGS approach using baits targeting most of the CoV species. Using this technology, we effectively reduced sequencing costs by increasing the sensitivity of detection. We discovered nine full genomes of bat CoVs in this study and revealed great genetic diversity for eight of them. IMPORTANCE Active surveillance is both urgent and essential to predict and mitigate the emergence of bat-origin CoV in humans and livestock. However, great genetic diversity increases the chance of homologous recombination among CoVs. Performing targeted PCR, a common practice for many surveillance studies, would not reflect this diversity. NGS, on the other hand, is an expensive methodology and is prone to missing low-abundance CoV sequences. Here, we employ a capture-based NGS approach using baits targeting all CoVs. Our work demonstrates that targeted, cost-effective, large-scale, genome-level surveillance of bat CoVs is now highly feasible. 2020. Discovery of bat coronaviruses through surveillance and probe capture-based next-generation sequencing. mSphere 5:e00807-19. https://doi .org/10.1128/mSphere.2020. Correction for Li et al., "Discovery of bat coronaviruses through surveillance and probe capture-based next-generation sequencing." mSphere 5: e00170-20. https://doi.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Discovery of Bat Coronaviruses through Surveillance and Probe Capture-Based Next-Generation Sequencing

Hao

Zhu

et al. 2020

mSphere

105

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“…It also enables the simultaneous detection of the genetic information of pathogens of all taxa including viruses, bacteria and eukaryotic pathogens like parasites or fungi [2]. However, poor sample quality, low pathogen loads and high levels of background consisting of nucleic acids of the host or accompanying bacteria often lead to a detrimental pathogen/background nucleic acid ratio of the final sequencing libraries [3]. Analyses of the resulting sequence datasets can be laborious and time consuming and difficult to interpret, even for experts.…”

Section: Introductionmentioning

confidence: 99%

Next-generation diagnostics: virus capture facilitates a sensitive viral diagnosis for epizootic and zoonotic pathogens including SARS-CoV-2

Wylezich

Calvelage

Schlottau

et al. 2020

Preprint

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AbstractBackgroundThe detection of pathogens in clinical and environmental samples using high-throughput sequencing (HTS) is often hampered by large amounts of background information, which is especially true for viruses with small genomes. Enormous sequencing depth can be necessary to compile sufficient information for identification of a certain pathogen. Generic HTS combining with in-solution capture enrichment can markedly increase the sensitivity for virus detection in complex diagnostic samples.MethodsA virus panel based on the principle of biotinylated RNA-baits was developed for specific capture enrichment of epizootic and zoonotic viruses (VirBaits). The VirBaits set was supplemented by a SARS-CoV-2 predesigned bait set for testing recent SARS-CoV-2 positive samples. Libraries generated from complex samples were sequenced via generic HTS and afterwards enriched with the VirBaits set. For validation, an internal proficiency test for emerging epizootic and zoonotic viruses (African swine fever virus, Ebolavirus, Marburgvirus, Nipah henipavirus, Rift Valley fever virus) was conducted.ResultsThe VirBaits set consists of 177,471 RNA-baits (80-mer) based on about 18,800 complete viral genomes targeting 35 epizootic and zoonotic viruses. In all tested samples, viruses with both DNA and RNA genomes were clearly enriched ranging from about 10-fold to 10,000-fold for viruses including distantly related viruses with at least 72% overall identity to viruses represented in the bait set. Viruses showing a lower overall identity (38% and 46%) to them were not enriched but could nonetheless be detected based on capturing conserved genome regions. The internal proficiency test supports the improved virus detection using the combination of HTS plus targeted enrichment but also point to the risk of carryover between samples.ConclusionsThe VirBaits approach showed a high diagnostic performance, also for distantly related viruses. The bait set is modular and expandable according to the favored diagnostics, health sector or research question. The risk of carryover needs to be taken into consideration. The application of the RNA-baits principle turned out to be user-friendly, and even non-experts (without sophisticated bioinformatics skills) can easily use the VirBait workflow. The rapid extension of the established VirBaits set adapted to actual outbreak events is possible without any problems as shown for SARS-CoV-2.

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Assembly of Long‐Adapter Single‐Strand Oligonucleotide (LASSO) Probes for Massively Parallel Capture of Kilobase Size DNA Targets

2021

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Genome DNA sequencing has become an affordable means to resolve questions about the genetic background of life. However, the biological functions of many DNA-encoded sequences are still relatively unknown. A highly scalable and cost-effective cloning method to select natural DNA targets from genomic templates is therefore urgently needed to enable rapid understanding of the biological products of genomes. One such method involves LASSO probes, which are long single-stranded DNA oligonucleotides designed with a universal adapter that is used to link two sequences that are complementary to a genomic target of interest. Through a pooled assembly method, LASSOs can be made for multiplex DNA capture. Herein, we describe a robust, efficient method to assemble LASSO probe libraries using a Cre-recombinase-mediated reaction and a protocol for multiplex genome target capture. The starting components are a pre-LASSO probe library comprising short DNA oligo pools designed in silico and an Escherichia coli plasmid (pLASSO) that incorporates the pre-LASSO library. Through internal recombination of pLASSO with its inserts, a mature LASSO library in final configuration can be made with high purity. Assembly of a LASSO probe library takes 4 days, and target capture can be performed in a single day. With an exponentially growing list of new genomes available for investigation, this method can enable the rapid production of ORFeome libraries for high-throughput screening to identify biological functions as a complementary approach to understand genome functional biology.

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Hybrid Capture-Based Next Generation Sequencing and Its Application to Human Infectious Diseases

Abstract: This review describes target-enrichment approaches followed by next generation sequencing and their recent application to the research and diagnostic field of modern and past infectious human diseases caused by viruses, bacteria, parasites and fungi.

Cited by 93 publications

References 82 publications

Discovery of Bat Coronaviruses through Surveillance and Probe Capture-Based Next-Generation Sequencing

Discovery of Bat Coronaviruses through Surveillance and Probe Capture-Based Next-Generation Sequencing

Next-generation diagnostics: virus capture facilitates a sensitive viral diagnosis for epizootic and zoonotic pathogens including SARS-CoV-2

Assembly of Long‐Adapter Single‐Strand Oligonucleotide (LASSO) Probes for Massively Parallel Capture of Kilobase Size DNA Targets

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