Coast-to-coast spread of SARS-CoV-2 in the United States revealed by genomic epidemiology

Joseph R Fauver; Mary E. Petrone; Emma B Hodcroft; Kayoko Shioda; Hanna Y Ehrlich; Alexander G. Watts; Chantal B.F. Vogels; Anderson F. Brito; Tara Alpert; Anthony Muyombwe; Jafar Razeq; Randy Downing; Nagarjuna R. Cheemarla; Anne L Wyllie; Chaney C. Kalinich; Isabel Ott; Josh Quick; Nicholas J. Loman; Karla M. Neugebauer; Alexander L. Greninger; Keith R. Jerome; Pavitra Roychoundhury; Hong Xie; Lasata Shrestha; Meei-Li Huang; Virginia E. Pitzer; Akiko Iwasaki; Saad B. Omer; Kamran Khan; Isaac Bogoch; Richard A. Martinello; Ellen F. Foxman; Marie-Louise Landry; Richard A Neher; Albert I Ko; Nathan D. Grubaugh

Samples for this study were collected during an early testing phase by the Connecticut State Department of Public Health or the Yale Clinical Virology Laboratory at the Yale School of Medicine. Nasopharyngeal swabs were collected from patients presenting with symptoms of SARS-CoV-2 infection at multiple medical centers in Connecticut. These patients are all Connecticut residents, but we do not have access to location data associated with each of these early SARS-CoV-2 genomes to avoid patient identification. Swabs were placed in virus transport media (BD Universal Viral Transport Medium) immediately upon collection. Samples (200 µL) were subjected to total nucleic acid extraction using the NUCLISENS easyMAG platform (BioMérieux, France) at the Yale Clinical Virology Laboratory. The recommended CDC RT-qPCR assay was used to test for the presence of SARS-CoV-2 RNA (Centers for Disease Control and Prevention, 2020c) . A total of 10 samples from 10 different individuals with cycle-threshold (CT) values less than 35 were selected to move forward with next generation sequencing (NGS). Of these, we were successfully able to generate sequencing libraries from nine samples. SARS-CoV-2 positive samples were processed for NGS using a highly multiplexed PCR amplicon approach for sequencing on the Oxford Nanopore Technologies (ONT; Oxford, United Kingdom) MinION using the V1 primer pools (Quick et al., 2017) . Sequencing libraries were barcoded and multiplexed using the Ligation Sequencing Kit and Native Barcoding Expansion pack (ONT) following the ARTIC Network's library preparation protocol (Quick, 2020) with the following minor modifications: cDNA was generated with SuperScriptIV VILO Master Mix (ThermoFisher Scientific, Waltham, MA, USA), a total of 20 ng of each sample was used as input into end repair, end repair incubation time was increased to 25 minutes followed by a 1:1 bead-based clean up, and Blunt/TA ligase (New England Biolabs, Ipswich, MA, USA) was used to ligate barcodes to each sample. cDNA synthesis and amplicon generation was performed concurrently for each sample. Samples were processed by CT value to reduce the likelihood of contamination from high titer samples to low titer samples. Barcoding, adaptor ligation, and sequencing was performed on samples with CT values between 25-35 (low titer group) prior to samples with CT values below 25 (high titer group) ( Data S1 ). Two samples, Yale-006 and Yale-007, were diluted 1:100 in nuclease-free water prior to cDNA synthesis. A no template control was created at the cDNA synthesis step and amplicon generation step to detect cross-contamination between samples. Controls were barcoded and sequenced with both the high and low titer sample groups.