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. 2020 Nov 27;12(12):1358.
doi: 10.3390/v12121358.

Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples

Leonard Schuele  1   2 , Hayley Cassidy  1 , Erley Lizarazo  1 , Katrin Strutzberg-Minder  3 , Sabine Schuetze  4 , Sandra Loebert  4 , Claudia Lambrecht  4 , Juergen Harlizius  4 , Alex W Friedrich  1 , Silke Peter  2 , Hubert G M Niesters  1 , John W A Rossen  1   5 , Natacha Couto  1   6
Affiliations

Assessment of Viral Targeted Sequence Capture Using Nanopore Sequencing Directly from Clinical Samples

Leonard Schuele et al. Viruses. .

Abstract

Shotgun metagenomic sequencing (SMg) enables the simultaneous detection and characterization of viruses in human, animal and environmental samples. However, lack of sensitivity still poses a challenge and may lead to poor detection and data acquisition for detailed analysis. To improve sensitivity, we assessed a broad scope targeted sequence capture (TSC) panel (ViroCap) in both human and animal samples. Moreover, we adjusted TSC for the Oxford Nanopore MinION and compared the performance to an SMg approach. TSC on the Illumina NextSeq served as the gold standard. Overall, TSC increased the viral read count significantly in challenging human samples, with the highest genome coverage achieved using the TSC on the MinION. TSC also improved the genome coverage and sequencing depth in clinically relevant viruses in the animal samples, such as influenza A virus. However, SMg was shown to be adequate for characterizing a highly diverse animal virome. TSC on the MinION was comparable to the NextSeq and can provide a valuable alternative, offering longer reads, portability and lower initial cost. Developing new viral enrichment approaches to detect and characterize significant human and animal viruses is essential for the One Health Initiative.

Keywords: next-generation sequencing; one health; porcine viruses; shotgun metagenomic sequencing; targeted sequence capture; viral metagenomics; virome.

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Conflict of interest statement

John W. A. Rossen is employed by IDbyDNA. Silke Peter consults for IDbyDNA. This did not have any influence on the interpretation of reviewed data and conclusions drawn nor on the drafting of the manuscript, and no support was obtained from them. All other authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Workflow of the laboratory procedures evaluated in this study. (a) Shotgun approach for a baseline on Oxford Nanopore Technologies (ONT) platforms; (b) ViroCap on pooled barcoded library pools using a 20 min and 20 h hybridization time; (c) ViroCap on individual samples using a 20 h hybridization time; (d) standard ViroCap protocol on Illumina platforms. Abbreviations: ONT, Oxford Nanopore Technologies; SISPA, Sequence-Independent Single-Primer-Amplification.
Figure 2
Figure 2
Sample composition with and without TSC on the ONT MinION and with TSC on the Illumina NextSeq. Abbreviations: M, MinION; MV, MinION with ViroCap; NV, NextSeq with ViroCap.
Figure 3
Figure 3
Visualization of the coverage depth for four clinically relevant viruses detected in our study. (a) Influenza A virus; (b) Porcine respirovirus 1; (c) Enterovirus D68; (d) Norovirus GII.4. Reads were not normalized and therefore actual depth was dependent on the number of total reads of the sample. * All 8 influenza A virus segments were concatenated into a single genome. Abbreviations: M, MinION; MV, MinION with ViroCap; NV, NextSeq with ViroCap; IAV, influenza A virus; PRV1, porcine respirovirus 1; EV-D68, enterovirus D68; NoV GII.4, norovirus GII.4.
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