key: cord-0703002-6ucdibzh
authors: Paden, Clinton R; Tao, Ying; Queen, Krista; Zhang, Jing; Li, Yan; Uehara, Anna; Tong, Suxiang
title: Rapid, sensitive, full genome sequencing of Severe Acute Respiratory Syndrome Virus Coronavirus 2 (SARS-CoV-2)
date: 2020-04-23
journal: bioRxiv
DOI: 10.1101/2020.04.22.055897
sha: ec8680250a3c8d3e975ef3bf13c17d3c637d488a
doc_id: 703002
cord_uid: 6ucdibzh

SARS-CoV-2 recently emerged, resulting a global pandemic. Rapid genomic information is critical to understanding transmission and pathogenesis. Here, we describe validated protocols for generating high-quality full-length genomes from primary samples. The first employs multiplex RT-PCR followed by MinION or MiSeq sequencing. The second uses singleplex, nested RT-PCR and Sanger sequencing.

In December 2019, SARS-CoV-2, the etiological agent of Coronavirus Disease 2019 18 (COVID-19), emerged in Wuhan, China. Since then it has rapidly spread to the rest of the world 19 (1-3). As of April 16, 2020, there have been 1,991,562 confirmed cases, including 130,885 20 deaths, in 185 countries or regions (4). 21 Initial sequencing of SARS-CoV-2 showed limited genetic variation between cases, but 22 did document specific changes that may be useful for understanding the source and transmission 23 chains (5-8). Because SARS-CoV-2 has shown the capacity to spread rapidly and lead to a range 24 of presentations in infected persons, from asymptomatic infection to mild, severe, or fatal 25 disease, it is important to identify genetic variants in order to understand any changes in 26 transmissibility, tropism, and pathogenicity. Sequence data can be used to inform decisions to 27 better manage the spread of disease. 28 In this report, we describe the design and use of two PCR-based methods for sequencing 29 SARS-CoV-2 clinical specimens. The first is a multiplex PCR panel followed by sequencing on On January 10, 2020, the first SARS-CoV-2 genome sequence was released online (11).

That day, we designed two complementary panels of primers to amplify the virus genome for 39 sequencing. For one panel, we used the PRIMAL primer design tool (9) to design multiplex To determine the sensitivity of each sequencing strategy, we generated a set of six ten- CoV-2 reads and >99% genome coverage at 20X depth, decreasing to an average of 93% 62 genome coverage at CT 33.2 and 48% at CT 35 ( Figure 1A and 1B). Further, we were able to 63 obtain full >20X genomes within the first 40-60 minutes of sequencing ( Figure 1C ). steps, the data approaches 100% consensus accuracy (Table 1) . Identical results were found 72 using the R9.4.1 pore, through the CT 33.2 samples (data not shown). We noted larger deletions 73 in some of the CT 33.2+ samples which likely reflect biases from limited copy numbers.

In the MiSeq data, we observed a similar trend in percent genome coverage at 100X 75 depth, and a slightly lower percent mapped reads, compared to Nanopore data ( Figure 1A while Sanger data was available the following day. 87 We used the multiplex PCR strategy in subsequent SARS-CoV-2 clinical cases (n=167), 88 ranging in CT values from 15.7 to 40 (mean 28.8, median 29.1). In cases below CT 33, we 89 observed an average of 99.02% specific reads and 99.2% genome coverage at >20X depth 90 (Figures 2A and 2B ). Between CT 30-33, genome coverage varied by sample, and declined The multiplex PCR strategy is effective at generating full genome sequences up to CT 33.

The singleplex, nested PCR is effective up to CT 35, varying based on sample quality. The 106 turnaround time for the multiplex PCR MinION protocol is about 8 hours from nucleic acid to 107 consensus sequence, compared to Sanger sequencing at about 14-18 hours (Table 2) . 108 Importantly, the multiplex PCR protocols offer an efficient, cost-effective, scalable system, and 109 add little time and complexity as sample numbers increase ( Table 2) . The results from this study 110 suggest multiplex PCR may be used effectively for routine sequencing, complemented by 111 singleplex, nested PCR for low virus-titer samples and confirmation sequencing. 

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