Abstract
Background: Nasopharyngeal samples contain higher quantities of bacterial and host nucleic acids relative to viruses; presenting challenges during virus metagenomics sequencing, which underpins agnostic sequencing protocols. We aimed to develop a viral enrichment protocol for unbiased whole-genome sequencing of respiratory syncytial virus (RSV) from nasopharyngeal samples using the Oxford Nanopore Technology (ONT) MinION platform. Methods: We assessed two protocols using RSV positive samples. Protocol 1 involved physical pre-treatment of samples by centrifugal processing before RNA extraction, while Protocol 2 entailed direct RNA extraction without prior enrichment. Concentrates from Protocol 1 and RNA extracts from Protocol 2 were each divided into two fractions; one was DNase treated while the other was not. RNA was then extracted from both concentrate fractions per sample and RNA from both protocols converted to cDNA, which was then amplified using the tagged Endoh primers through Sequence-Independent Single-Primer Amplification (SISPA) approach, a library prepared, and sequencing done. Statistical significance during analysis was tested using the Wilcoxon signed-rank test. Results: DNase-treated fractions from both protocols recorded significantly reduced host and bacterial contamination unlike the untreated fractions (in each protocol p<0.01). Additionally, DNase treatment after RNA extraction (Protocol 2) enhanced host and bacterial read reduction compared to when done before (Protocol 1). However, neither protocol yielded whole RSV genomes. Sequenced reads mapped to parts of the nucleoprotein (N gene) and polymerase complex (L gene) from Protocol 1 and 2, respectively. Conclusions: DNase treatment was most effective in reducing host and bacterial contamination, but its effectiveness improved if done after RNA extraction than before. We attribute the incomplete genome segments to amplification biases resulting from the use of short length random sequence (6 bases) in tagged Endoh primers. Increasing the length of the random nucleotides from six hexamers to nine or 12 in future studies may reduce the coverage biases.
Highlights
Unbiased sequencing of bacterial, fungal and viral communities has been used to characterize the microbial diversity in nasopharyngeal samples and aid in explaining diseases of unknown aetiologies (Camelo-Castillo et al, 2019; Geliebter et al, 2020; Lu et al, 2020)
Though Sequence-Independent Single-Primer Amplification (SISPA) has previously proved effective in metagenomics studies, it results in preferential sequencing of the most abundant nucleic acid material in a nasopharyngeal sample; mainly host and bacteria (Djikeng et al, 2008; Goya et al, 2018)
For samples to be included in this study, they had to have been confirmed positive for respiratory syncytial virus (RSV) using the indirect immunoflourescent antibody test (IFAT) and reverse transcription polymerase chain reaction (RT-PCR) method and recorded high viral load as identified by low cycle threshold scores (Ct < 24)
Summary
Fungal and viral communities has been used to characterize the microbial diversity in nasopharyngeal samples and aid in explaining diseases of unknown aetiologies (Camelo-Castillo et al, 2019; Geliebter et al, 2020; Lu et al, 2020). Though SISPA has previously proved effective in metagenomics studies, it results in preferential sequencing of the most abundant nucleic acid material in a nasopharyngeal sample; mainly host and bacteria (Djikeng et al, 2008; Goya et al, 2018). SISPA, centrifugal filtration and DNase treatment were employed in several studies (Chrzastek et al, 2017; Goya et al, 2018; Lewandowski et al, 2020) and deemed effective in enhancing viral read representation and in reducing bacterial and host contamination. We aimed to develop a viral enrichment protocol for unbiased whole-genome sequencing of respiratory syncytial virus (RSV) from nasopharyngeal samples using the Oxford Nanopore Technology (ONT) MinION platform. DNase treatment after RNA extraction (Protocol 2) enhanced host and bacterial read reduction compared to when done before (Protocol 1). Sequenced reads mapped to parts of the nucleoprotein (N gene) and polymerase complex (L gene) from Protocol 1 and 2, respectively
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
