Nanopore Targeted Sequencing for the Accurate and Comprehensive Detection of SARS-CoV-2 and Other Respiratory Viruses

Abstract

The ongoing global novel coronavirus pneumonia COVID-19 outbreak has engendered numerous cases of infection and death. COVID-19 diagnosis relies upon nucleic acid detection; however, currently recommended methods exhibit high false-negative rates and are unable to identify other respiratory virus infections, thereby resulting in patient misdiagnosis and impeding epidemic containment. Combining the advantages of targeted amplification and long-read, real-time nanopore sequencing, herein, nanopore targeted sequencing (NTS) is developed to detect SARS-CoV-2 and other respiratory viruses simultaneously within 6-10 h, with a limit of detection of ten standard plasmid copies per reaction. Compared with its specificity for five common respiratory viruses, the specificity of NTS for SARS-CoV-2 reaches 100%. Parallel testing with approved real-time reverse transcription-polymerase chain reaction kits for SARS-CoV-2 and NTS using 61 nucleic acid samples from suspected COVID-19 cases show that NTS identifies more infected patients (22/61) as positive, while also effectively monitoring for mutated nucleic acid sequences, categorizing types of SARS-CoV-2, and detecting other respiratory viruses in the test sample. NTS is thus suitable for COVID-19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses and pathogens.

Keywords: COVID-19; SARS-CoV-2; nanopore sequencing; respiratory virus; targeted amplification.

Figure 1

Amplification targets of the NTS and RT‐qPCR method. NTS detected 12 fragments including ORF1ab and virulence factor‐encoding regions. For RT‐qPCR, the Chinese CDC recommends orf1ab and N sites as targets,^{[ 51 ]} the United States CDC recommends three target sites in the N gene,^{[ 52 ]} and literature recommend RNA‐dependent RNA polymerase (RdRP) in orf1ab and E sites as the targets.^{[ 53 ]} Kit 1 is a CFDA‐approved kit with two target sites used in this study; kit 2 is a CFDA‐approved kit with three target sites used in this study.

Figure 2

Performance verification test of NTS for detecting SARS‐CoV‐2 using standard synthetic S and N genes. Comparison of all SARS‐CoV‐2 reads detected by NTS in replicates with different concentrations and negative controls using 10 min (a) or 1 h (b) sequencing data. Read counts mapped to each target region of the SARS‐CoV‐2 genome in replicates with different concentrations with 10 min (c) to 1 h (d) sequencing data. Two‐tailed Student t‐test (for normal distribution samples) or Mann–Whitney U‐test (for non‐normal distribution samples): ns, not significant, *p < 0.05; bars represent the means ± SD.

Figure 3

Turnaround time of NTS. The total nucleic acids, including single‐stranded DNA/RNA and double‐stranded DNA, were extracted, and the total RNA in the total nucleic acids was reverse transcript to cDNA. Specific regions of the DNA virus and cDNA of the RNA virus were amplified by multiplex PCR (one tube for SARS‐CoV‐2 and another tube for respiratory viruses). Next, the same barcode was added to both ends of the PCR product from the same sample using a barcoding PCR step. The barcoded products of each sample were pooled and used for sequencing library preparation. The barcoding PCR step in the red frame can be removed by directly ligating the barcode to products of multiplex PCR during library preparation using a commercial kit, the turnaround time and risk of cross‐contamination could be further reduced. Time for bioinformatics analysis depends on data size and the computer's performance.

Figure 4

Specificity test. Five throat samples containing influenza A virus, influenza B virus, parainfluenza, respiratory syncytial virus, and rhinovirus were selected to test the cross‐reactivity of the SARS‐CoV‐2 primer panel for common respiratory viruses in duplicate. TE buffer spiked with human DNA was parallelly tested as a negative control. None of the sequencing reads could be correctly mapped to the SARS‐CoV‐2 genome in all samples and the negative control. Nonviral reads could not correctly be mapped to any reference in the viral genome database, which may derive from the nonspecific amplification of human genome. Several sequencing reads in samples could be mapped to other virus genomes.

Figure 5

NTS testing in a front‐line hospital in Wuhan.

Figure 6

Comparison of 61 nucleic acid samples from clinical samples obtained using NTS (4 h) and RT‐qPCR. a) Comparison of 45 nucleic acid samples from samples of patients with suspected COVID‐19 obtained using NTS and RT‐qPCR (kit 2). b) Comparison of 16 nucleic acid samples from patients with confirmed disease obtained using NTS and RT‐qPCR (kit 1). The numbers in the table on the left represent the number of mapped reads according to our rules. PC: positive control. The plasmid harboring an S gene was used as a positive control in NTS testing; a positive sample in the kit served as a positive control in RT‐qPCR testing. NC: negative control. TE buffer was used as a negative control in NTS testing; H₂O in the kit served as a negative control in RT‐qPCR testing. All positive sample and negative sample in NTS were introduced from nucleic acid extraction. Pos: positive. Inc: inconclusive. Neg: Negative.

Figure 7

Genome wide SNP analysis. a) Allele frequency of 50 NTS positive samples (top) and 1145 samples from GISAID (bottom). The altered nucleotides are colored with four different colors. We removed samples with uncertain nucleotides when calculating allele frequency of the 50 NTS positive samples for each SNP (Experimental Section). b) A part of the LD plot contained the most high D value mutation pairs. Mutations G28077C and C24034T have the highest D value. c) Typing result of 31 NTS positive samples.