Sequencing of the positive coronavirus specimens
Sanger sequencing of bat coronavirus: The RT-PCR products were separated on 1.5 per cent agarose gel, and 440 bp bands were excised. The excised gels were extracted and purified using a QIAQuick gel extraction kit (Qiagen, Hilden, Germany). The purified products were quantified, and chain-terminated PCR reactions were performed using pathogen-specific forward and reverse primers21 with the BigDye Terminator 3.1 sequencing kit (Applied Biosystems, USA). BigDye reactions were purified using the DyeEx 2.0 spin kit (Qiagen, Germany). The purified chain-terminated reactions were sequenced using the ABI PRISM® 3100 Automated DNA Sequencer (Thermo Fisher Scientific, USA). The sequence data generated were assembled using the Sequencer 5.1 software (Accelrys Inc., USA).
Next-generation sequencing (NGS) of bat coronavirus: Selected bat specimens were used for RNA extraction2223. RNA libraries were prepared and quantified by Qubit® 2.0 Fluorometer (Invitrogen, USA). NEB NextrRNA depletion kit (New England Biolabs, USA) was used to remove host ribosomal RNA and re-quantified using Qubit® 2.0 Fluorometer (Invitrogen, USA). In brief, the RNA library preparation involved fragmentation, adenylation, adapter ligation and amplification. The amplified libraries were quantified using KAPA Library Quantification Kit (KapaBiosystems, Roche, Switzerland) as per the manufacturer's protocol and further loaded onto the Illumina Miniseq NGS platform (Illumina, USA)2223.
The FASTQ files generated after the completion of the run were analyzed using CLC Genomics Workbench software version 11 (CLC, Qiagen, Germany). De novo assembly programme was used to assemble contiguous sequences (contigs). The contigs generated were analyzed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to identify matching sequences. The closest matching sequence from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) was used for reference mapping.
Phylogenetic analysis of partial and complete genome sequences of bat coronavirus: The CoV sequences retrieved from RS specimens of Rousettus spp. bats (n=4) were aligned with whole-genome sequences from GenBank using the create alignment function of the CLC genomics workbench (https://digitalinsights.qiagen.com). Partial RdRp gene sequences (~419 bp) retrieved by Sanger sequencing, for both the bat species specimens (genomic location: 14,701-15,120) were used to construct a phylogenetic tree along with the available RdRp sequences in GenBank. Phylogenetic analysis was carried out using the neighbour-joining method available in MEGA v7 software24 using the Kimura 2-parameter nucleotide (nt) substitution model with 1000 bootstrap replicates. The nt divergence for the open reading frame (ORF) 1a polyprotein (ORF 1a), ORF 1b polyprotein (ORF 1b), spike protein (S), nucleocapsid phospoprotein (N), envelope protein (E) and membrane glycoprotein (M) genes was estimated using the Kimura 2-parameter model as implemented in the MEGA software. The sequences retrieved in the current study, along with those downloaded from GenBank, were grouped into the genus.
The viruses from the β-CoV genus were further grouped into lineages, L_A, L_B, L_C and L_D, to estimate the evolutionary divergence over the respective gene sequence pairs between groups using the MEGA software24. The distance was estimated using a Kimura 2-parameter model with uniform rates among the sites. The bootstrap of 500 replicates was used to estimate the variation in the model.