Metagenomic Nanopore Sequencing of Influenza Virus Direct from Clinical Respiratory Samples

Abstract

Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. We therefore set out to apply the Oxford Nanopore Technologies sequencing method to metagenomic sequencing of respiratory samples. We generated influenza virus reads down to a limit of detection of 10 2 to 10 3 genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads (P ϭ 4.7 ϫ 10 Ϫ5 ). Applying our methods to clinical throat swabs, we generated influenza virus reads for 27/27 samples with mid-to-high viral titers (cycle threshold [C T ] values, Ͻ30) and 6/13 samples with low viral titers (C T values, 30 to 40). No false-positive reads were generated from 10 influenza virus-negative samples. Thus, Nanopore sequencing operated with 83% sensitivity (95% confidence interval [CI], 67 to 93%) and 100% specificity (95% CI, 69 to 100%) compared to the current diagnostic standard. Coverage of full-length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza virus titers, we were able to reconstruct Ͼ99% complete sequences for all eight gene segments. We also detected a human coronavirus coinfection in one clinical sample. While further optimization is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses.

Characterization of Emerging Swine Viral Diseases through Oxford Nanopore Sequencing Using Senecavirus A as a Model

“…From this study, an advantage of direct RNA sequencing over amplicon sequencing, such as PCR-cDNA, was that DRS showed a quantitative relationship between input viral titers and output sequencing reads. Similarly, a strong relationship between influenza viral titers and influenza sequencing reads using Oxford Nanopore direct RNA sequencing technology was observed by other research groups [ 57 ]. However, in a hepatitis B virus (HBV) study using Oxford Nanopore amplicon sequencing (which includes a PCR step similar to our PCS protocol), considerable variability in total yields and the proportion of mapped HBV reads between sequencing runs was observed concluding that it was not quantitative [ 59 ].…”

Field-Adapted Full Genome Sequencing of Peste-Des-Petits-Ruminants Virus Using Nanopore Sequencing

“…By comparing, the consensus sequences produced by the described protocol with previously published sequences produced using the other sequencing techniques; we were able to evaluate the performance of the protocol. Other comparisons of the minION sequencing technique to other more traditional, and labor and equipment intensive have equally found that the method produces high quality sequences (29).…”

Isolation and rapid sharing of the 2019 novel coronavirus ( SARS ‐CoV‐2) from the first patient diagnosed with COVID ‐19 in Australia

“…We extracted RNA for whole genome sequencing of the viral isolate. Briefly, RNA was extracted from clarified cell culture supernatant and randomly amplified cDNA prepared by sequence‐independent single‐primer amplification (SISPA) . Sequencing was performed with a combination of Oxford Nanopore Technologies and Illumina short‐read sequencing.…”