High throughput nanopore sequencing of SARS-CoV-2 viral genomes from patient samples

Abstract

In late 2019, a novel coronavirus began spreading in Wuhan, China, causing a potentially lethal respiratory viral infection. By early 2020, the novel coronavirus, called SARS-CoV-2, had spread globally, causing the COVID-19 pandemic. The infection and mutation rates of SARS-CoV-2 make it amenable to tracking introduction, spread and evolution by viral genome sequencing. Efforts to develop effective public health policies, therapeutics, or vaccines to treat or prevent COVID-19 are also expected to benefit from tracking mutations of the SARS-CoV-2 virus. Here we describe a set of comprehensive working protocols, from viral RNA extraction to analysis using established visualization tools, for high throughput sequencing of SARS-CoV-2 viral genomes using a MinION instrument. This set of protocols should serve as a reliable "how-to" reference for generating quality SARS-CoV-2 genome sequences with ARTIC primer sets and long-read nanopore sequencing technology. In addition, many of the preparation, quality control, and analysis steps will be generally applicable to other sequencing platforms.

Keywords: COVID-19; MinION; SARS-CoV-2; genome; nanopore; sequencing.

Figure 1.

Workflow for SARS-CoV-2 viral genome sequencing with a MinION instrument.

Figure 2.

Representative qPCR and ARTIC PCR and genome sequencing coverage.A. Amplification curves from SARS-CoV-2 qPCR assay and IDT 2019-nCoV CDC approved N2 primers and probes. Baseline adjustment was manually set to 200. B. Representative agarose gel electrophoresis of ~400 bp PCR products amplified using ARTIC nCoV V3 Primers (IDT). Reaction pool 1 (labels with suffixes -1) and pool 2 (labels with suffixes -2) were run side by side with 5 μl of sample on the gel. PBS (no template) extraction controls for pool 1 and 2 show no contamination. C. Coverage plot with amplicon 74 dropout using ARTIC V3 Primers (bottom panel). The plot shows no spike-in (red), 1× (15 nM) (blue), 3× (45 nM) (orange) and 5x (75 nM) (green) of 74_Right and 74_Left for sample 848 (C_t = 20.68) with coverage for amplicon 74 of 18×, 126×, 929× and 541×, respectively. Experiments were conducted at the same time using identical reagents and protocols. Amplicon 74 region is highlighted in yellow.

Figure 3.

Loading and running the MinION for SARS-CoV-2 genome sequencing.A. Image of the R9.4.1 Flow Cell displaying the different components of the device (left panel). An illustration demonstrating the priming port opening to primer port cover turning clockwise to expose the opening of the priming port (right panel). B. Example of RAMPART display. Top from left to right: (1) Top left shows coverage across the genomes for all samples. (2) Number of mapped reads through time. (3) Number of reads mapped for each barcode sample. (4) Heatmap showing reads mapped to nCoV2019| Wuhan-Hu-1 (accession MN908947). Bottom from left to right: (5) The coverage across the genome for an individual sample. (6) Read length distribution for an individual barcoded sample showing the expected peak of ~400 bp. (7) Percent > 20×, > 100×, and > 200× as a function of time.

Figure 4.

Nextstrain phylogenetic visualization and map view of SARS-CoV-2 genome sequences.A. Phylogenetic tree generated by the Nextstrain pipeline. Sequences are derived from samples taken in Illinois, from April 2020 through August 2021, and sequenced by our laboratory. Clade colors are indicated. B. Map of Illinois showing sample locations, by county. The size of the circle indicates the relative number of sequences derived from that county. The pie chart indicates the proportional distribution of Nextstrain clades at that location. Clade designation colors correlate with those designated in panel (A).