Nanopore Metagenomic Sequencing for Detection and Characterization of SARS-CoV-2 in Clinical Samples

Abstract

The COVID-19 pandemic has underscored the need for rapid novel diagnostic strategies to detect and characterize pathogens from clinical specimens. The MinION sequencing device allows for rapid, cost-effective, high-throughput sequencing; useful features for translation to clinical laboratory settings. Metagenomic Next-Generation Sequencing (mNGS) approaches provide the opportunity to examine the entire genomic material of a sample; allowing for detection of emerging and clinically relevant pathogens that may be missed in targeted assays. Here we present a pilot study on the performance of Sequence-Independent Single Primer Amplification (SISPA) to amplify RNA randomly for the detection and characterization of SARS-CoV-2. We designed a classifier that corrects for barcode crosstalk between specimens. Our assay yielded 100% specificity overall and 95.2% sensitivity for specimens with a RT-qPCR cycle threshold value less than 30. We assembled 10 complete (>95% coverage at 20x depth), and one near-complete (>80% coverage at 20x depth) genomes from 20 specimens that were classified as positive by mNGS. We characterized these genomes through phylogenetic analysis and found that 10/11 specimens from British Columbia had a closest relative to another British Columbian specimen. Of five samples that we had both assembled genomes, as well as Variant of Concern (VOC) PCR results, we found 100% concordance between these results. Additionally, our assay was able to distinguish between the Alpha and Gamma variants, which was not possible with our VOC PCR technique. This study supports future work examining the broader feasibility of SISPA as a diagnostic strategy for the detection and characterization of viral pathogens.

BugSplit: highly accurate taxonomic binning of metagenomic assemblies enables genome-resolved metagenomics

“…For the detection of an emerging coronavirus, nasopharyngeal swabs (n = 2) were collected as part of routine testing at Vancouver General Hospital during Fall 2020 ( ORF1ab C t values = 14.7, 20.6) and cultured SARS-CoV-2 viral particles ( RdRp C t value = 18.3) were obtained from the BC Centre for Disease Control Public Health Laboratory. Both clinical samples and cultured virions were extracted and randomly amplified through sequence-independent single-primer amplification as previously described 60 . Samples were sequenced on Oxford Nanopore MinION devices and basecalling was performed with Guppy (Oxford Nanopore Technologies).…”

Machine-learning based detection of adventitious microbes in T-cell therapy cultures using long read sequencing

“…Long read MinION sequencing offers a rapid, simple-setup, real-time reads (contaminant identification before sequencing completion) and low-cost approach to DNA sequencing. 11,12 Compared to Illumina platforms, which can take days to weeks to complete sequencing and bioinformatics analyses, the Nanopore sequencing can provide results in less than 24 h. 13,14 Another advantage with the long read MinION sequencer, is greater taxonomic resolution than amplicon sequencing using the Illumina MiSeq system at the species level. 15 This positions an amplicon sequencing approach using the long read MinION combined with a full size metagenome database favourably for managing rapid release testing.…”