Rapid viral metagenomics using SMART-9N amplification and nanopore sequencing

Abstract

Emerging and re-emerging viruses are a global health concern. Genome sequencing as an approach for monitoring circulating viruses is currently hampered by complex and expensive methods. Untargeted, metagenomic nanopore sequencing can provide genomic information to identify pathogens, prepare for or even prevent outbreaks. SMART (Switching Mechanism at the 5′ end of RNA Template) is a popular method for RNA-Seq but most current methods rely on oligo-dT priming to target polyadenylated mRNA molecules. We have developed two random primed SMART-Seq approaches, ‘SMART-9N’, and a version compatible with barcoded PCR primers available from Oxford Nanopore Technologies, ‘Rapid SMART-9N’, for the detection, characterization, and whole-genome sequencing of RNA viruses. The methods were developed using viral isolates, clinical samples, and compared to a gold-standard amplicon-based method. From a Zika virus isolate the SMART-9N approach recovered 10kb of the 10.8kb RNA genome in a single nanopore read. We also obtained full genome coverage at a high depth coverage using the Rapid SMART-9N, which takes only 10 minutes and costs up to 45% less than other methods. We found the limits of detection of these methods to be 6e00 focus forming units (FFU)/mL with 99.02% and 87.58% genome coverage for SMART-9N and Rapid SMART-9N respectively. Yellow fever virus plasma samples and SARS-CoV-2 nasopharyngeal samples previously confirmed by RT-qPCR with a broad range of Ct-values were selected for validation. Both methods produced greater genome coverage when compared to the multiplex PCR approach and we obtained the longest single read of this study (18.5 kb) with a SARS-CoV-2 clinical sample, 60% of the virus genome using the Rapid SMART-9N method. This work demonstrates that SMART-9N and Rapid SMART-9N are sensitive, low input, and long-read compatible alternatives for RNA virus detection and genome sequencing and Rapid SMART-9N improves the cost, time, and complexity of laboratory work.

Shotgun metagenomic sequencing of the first case of monkeypox virus in Brazil, 2022

“…DNA was quantified using fluorimetry with the Qubit dsDNA High Sensitivity Assay (Cat Nº Q32854, Life Technologies, Waltham, USA) on the Qubit 3.0 instrument (Life Technologies, USA). Shotgun metagenomics was performed using 10 ng of the extracted DNA and the Rapid PCR Barcoding kit (SQK-RPB004) -Oxford Nanopore Technologies (ONT, UK), adapted from the Rapid SMART-9N protocol 8 . PCR products were then purified using a 1:1 ratio of AMPure XP beads (Cat Nº A63881, Beckman Coulter, UK) and quantified.…”

Identification and characterization of the anti-SARS-CoV-2 activity of cationic amphiphilic steroidal compounds

“…SARS-CoV-2 genome sequencing was carried out using the MinION sequencing platform (Oxford Nanopore Technologies, https://nanoporetech.com ) and the ARTIC version 3 protocol ( https://nanoporetech.com ) and metagenomic using SMART-9N amplification using with MinION sequencing (Oxford Nanopore Technologies, Oxford, UK), as described elsewhere [ 11 ]. A multiplex PCR approach was used as previously described [ 12 , 13 ] for whole-genome amplification, using ARTIC V3 primer schemes.…”

Venezuelan equine encephalitis complex, Madariaga and Eastern equine encephalitis viruses genome detection in human and mosquito populations

“…To confirm virus species, subtype and genotype, we sequenced seven selected VEEV complex rRT-PCR positive mosquito and human samples from 2015 and 2022 using SMART-9N metagenomic sequencing ad previously described (28). In brief, viral RNA was treated with TURBO DNase (Thermo Fisher Scientific, USA) and concentrated with Zymo RNA clean & concentrator-5 (Zymo Research, USA) following protocol instructions.…”

“…In brief, viral RNA was treated with TURBO DNase (Thermo Fisher Scientific, USA) and concentrated with Zymo RNA clean & concentrator-5 (Zymo Research, USA) following protocol instructions. cDNA synthesis and PCR was performed as described (28). Fifty ng of the quantified PCR products were pooled using EXP-NBD104 (1-12) and EXP-NBD114 (13-24) Native Barcoding Kits (ONT, UK).…”