Rapid, Sensitive, Full-Genome Sequencing of Severe Acute Respiratory Syndrome Coronavirus 2

Abstract

We describe validated protocols for generating high-quality, full-length severe acute respiratory syndrome coronavirus 2 genomes from primary samples. One protocol uses multiplex reverse transcription PCR, followed by MinION or MiSeq sequencing; the other uses singleplex, nested reverse transcription PCR and Sanger sequencing. These protocols enable sensitive virus sequencing in different laboratory environments.

Keywords: COVID-19; SARS-CoV-2; coronavirus; coronavirus disease; genomics; high-throughput nucleotide sequencing; respiratory infections; severe acute respiratory syndrome coronavirus 2; viruses; whole-genome sequencing; zoonoses.

Figure 1

Limits of detection for sequencing severe acute respiratory syndrome coronavirus 2. Triplicate serial dilutions of virus isolate A12 (8) were amplified by using the singleplex or multiplex primer set. Multiplex amplicons were barcoded, library-prepped, and sequenced on an Oxford MinION apparatus (https://nanoporetech.com) or an Illumina MiSeq apparatus (https://www.illumina.com). A) Percentage of reads that map to the virus genome for each sample. B) Percentage of virus genome that is covered at >20× depth by the multiplex amplicons on the MinION (black) or >100× depth on the MiSeq (orange), or covered by the nested, singleplex amplicons (gray) (measured by presence or absence on a gel). C) Real-time analysis of MinION sequencing data. Each data point represents the average 20× genome coverage of three replicates. NTC, nontemplate controls (human cell nucleic acid carried through the PCR and library preparation). Asterisk (*) indicates that samples were not analyzed at that dilution.

Figure 2

Sequencing of severe acute respiratory syndrome coronavirus 2 clinical samples. A, B) Percentage mapped (A) and percentage genome coverage (B) for 167 clinical severe acute respiratory syndrome coronavirus 2 samples amplified by using a multiplex PCR strategy and sequenced on the MinION apparatus (https://nanoporetech.com). C) Time-lapse of 20× genome coverage obtained for clinical specimens at the indicated cycle threshold values. Data points indicate average coverage over time for various samples and grouped by run and the indicated C_t values. C_t, cycle threshold; N2, nucleoprotein 2.