BGI sequencing strategy

All collected samples were sent to BGI for sequencing. 140 μL bronchoalveolar lavage fluid samples (WH01 to WH04) were reserved for RNA extraction using the QIAamp Viral RNA Mini Kit (52904; Qiagen, Heiden, Germany), according to the manufacturer's recommendations. A probe-captured technique was used to remove human nucleic acid. The remaining RNA was reverse-transcribed into cDNA, followed by the second-strand synthesis. Using the synthetic double-stranded DNA, a DNA library was constructed through DNA-fragmentation, end-repair, adaptor-ligation, and PCR amplification. The constructed library was qualified with an Invitrogen Qubit 2.0 Fluorometer (ThermoFisher, Foster City, CA, USA), and the qualified double-stranded DNA library was transformed into a single-stranded circular DNA library through DNA-denaturation and circularisation. DNA nanoballs were generated from single-stranded circular DNA by rolling circle amplification, then qualified with Qubit 2.0 and loaded onto the flow cell and sequenced with PE100 on the DNBSEQ-T7 platform (MGI, Shenzhen, China).

After removing adapter, low-quality, and low-complexity reads, high-quality genome sequencing data were generated. Sequence reads were first filtered against the human reference genome (hg19) using Burrows-Wheeler Alignment.¹⁵ The remaining data were then aligned to the local nucleotide database (using Burrows-Wheeler Alignment) and non-redundant protein database (using RapSearch),¹⁶ downloaded from the US National Center for Biotechnology Information website, which contain only coronaviruses that have been published. Finally, the mapped reads were assembled with SPAdes¹⁷ to obtain a high-quality coronavirus genome sequence.

Primers were designed with use of OLIGO Primer Analysis Software version 6.44 on the basis of the assembled partial genome, and were verified by Primer-Blast (for more details on primer sequencs used please contact the corresponding author). PCR was set up as follows: 4·5 μL of 10X buffer, 4 μL of dNTP mix (2·5 μmol/L), 1 μL of each primer (10 μmol/L), and 0·75 units of HS Ex Taq (Takara Biomedical Technology, Beijing, China), in a total volume of 30 μL. The cDNAs reverse transcribed from clinical samples were used as templates, and random primers were used. The following program was run on the thermocycler: 95°C for 5 min; 40 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min as determined by product size; 72°C for 7 min; and a 4°C hold. Finally, the PCR products were separated by agarose gel electrophoresis, and products of the expected size were sequenced from both ends on the Applied Biosystems 3730 DNA Analyzer platform (Applied Biosystems, Life Technologies, Foster City, CA, USA; for more details on expected size please contact the corresponding author).