A Genomic Survey of SARS-CoV-2 Reveals Multiple Introductions into Northern California without a Predominant Lineage

Xianding Deng; Wei Gu; Scot Federman; Louis Du Plessis; Oliver Pybus; Nuno Faria; Candace Wang; Guixia Yu; Chao-Yang Pan; Hugo Guevara; Alicia Sotomayor-Gonzalez; Kelsey Zorn; Allan Gopez; Venice Servellita; Elaine Hsu; Steve Miller; Trevor Bedford; Alexander Greninger; Pavitra Roychoudhury; Michael Famulare; Helen Y Chu; Jay Shendure; Lea Starita; Catie Anderson; Karthik Gangavarapu; Mark Zeller; Emily Spencer; Kristian Andersen; Duncan MacCannell; Suxiang Tong; Gregory Armstrong; Clinton Paden; Yan Li; Ying Zhang; Scott Morrow; Matthew Willis; Bela Matyas; Sundari Mase; Olivia Kasirye; Maggie Park; Curtis Chan; Alexander Yu; Shua Chai; Elsa Villarino; Brandon Bonin; Debra Wadford; Charles Y Chiu

The extracted RNA described above was converted to cDNA using MSSPE method (Metagenomic Sequencing with Spiked Primer Enrichment) as previously described (15) . The custom designed SARS-CoV-2 primers (13-nucleotide in length, IDT Technologies) were constructed using 30 early SARS-CoV-2 genome references in the National Instiutes of Health (NIH) GenBank database, and were subsequently added to the reverse transcription process (Table S1 ). The cDNA eluate was made into sequencing libraries through tagmentation (Nextera XT, Illumina), and were individually barcoded in preparation for Illumina sequencing. The samples were then bead-washed with AMPure XP beads (Beckman Coulter, Brea, USA) at 0.92X (23uL). The barcoded library then directly proceeds into a library recovery step. 5uL of the library is added to an amplification reaction mix (10uL Phusion 5X Buffer (ThermoFisher Scientific, Waltham, USA), 2.5uL of 10uM forward and reverse general primers, 1uL of 12.5mM dNTPs, 0.5uL Phusion enzyme (ThermoFisher Scientific, Waltham, USA), 31uL nuclease-free water) for 14 cycles, with cycling parameters as previously described (22) . The amplicons were again purified using 0.9X volume of AMPure XP beads. At this point, the purified PCR product can be analyzed by gel electrophoresis to check library size.The eluted library was quantified using a Qubit fluorometer. Sequencing libraries were then sequenced on MiSeq, Nextseq, or HiSeq 1500 (Illumina Inc., San Diego, USA) as 1x150 single-end or 2x150 paired-end reads.