COVID-19 laboratory screening
Nasopharyngeal/Nasal/Oropharyngeal swabs in viral transport medium (VTM) received from acute phase patients with defined symptoms, asymptomatic cases with contact history with positive patients/ travel history were processed for laboratory confirmation of SARS-CoV-2 at Defence Research and Development Establishment, (DRDE) Gwalior, M.P., India. These samples were referred for molecular diagnosis from 10 different districts health authorities of Madhya Pradesh, India during the period of March –May 2020 of COVID-19 pandemic. Samples were anonymized and de-identified so researchers were blind to the identity of the patients. Only secondary non-identifying data including age, sex, and travel history were provided. The experimental protocols of this study were approved by Institutional Biosafety Committee (IBSC) vide no IBSC/VIRO-04/20/PKD. Ethical approval and informed consent waiver for this study has also been granted vide no. VCH/VEC/June-2021/04 of Vidya Ethics Committee, Gwalior, India. All methods were performed in accordance with the relevant guidelines and regulations.
Isolation of viral RNA
The samples were processed in a BSL-3 facility (High Containment Facility) following biosafety precautions at DRDE, Gwalior. The viral RNA was extracted from the referred samples using QIAamp viral RNA mini kit (Qiagen, Germany) as per manufacturer’s instructions. Briefly, 560 µl of lysis buffer was added to 140 µl of clinical specimen and incubated for 10 min at room temperature. The sample was passed through silica column followed by washing in 500 µl wash buffer and finally the viral RNA was eluted in 50 µl of elution buffer in a nuclease free tube and used as template in downstream molecular detection assay.
Laboratory investigation of COVID-19
Presence of SARS-CoV-2 was investigated by performing SARS-CoV-2 screening (Envelope, Human Rnase P genes) and confirmatory assays targeting (RdRP, ORF 1ab, N genes) to ensure RNA quality during sample collection as per WHO protocol24. The Taqman qRT-PCR was performed with dual labeled probes with reporter dyes FAM/VIC (E/Rnase P- Screening Assay), FAM/VIC (RdRP/ORF- Confirmatory Assay), Cy5 (N gene- Confirmatory assay). The fluorescence signals were recorded in ABI 7500 Dx Real time PCR (ABI, USA).
SARS-CoV-2 whole genome sequencing
The representative positive cases were selected on the basis of patients with travel history in the region (n = 6), patients with different age group from similar contact history (n = 10); introduction links from different areas to different families (n = 4) named as index case A(Shivpuri), index case B (Morena), index case C (Sheopur), index case D (Gwalior) COVID-19 positive death cases (n = 2) and index cases from different districts (n = 4). Based on above mentioned criteria, qRT-PCR positive cases (n = 26) were selected for whole genome sequencing. Briefly cDNA was synthesized using Superscript IV reverse transcriptase (NEB, USA) along with 23 mer oligo dT primer, 50 μM random hexamer and 10 mM dNTPs mix. The reaction was performed at 65 °C for 5 min and snap chilled on ice, Further reverse transcription was performed at 42 °C 50 min followed by 70 °C 10 min. The cDNA was stored at − 80 °C until further use.
Amplification of whole genome by Oxford nanopore platform
SARS-CoV-2 whole genome sequencing was performed as per ARCTIC protocol by Josh Quick. Briefly, cDNA from samples were used as template for multiplex PCR to amplify SARS-CoV-2 genome and the amplified products were purified. Purified cDNA amplicons from each sample were barcoded individually and pooled at equimolar concentration to prepare nanopore library. The quality and quantity of the pooled library was assessed using standard methods and sequenced on GridION-X5 nanopore sequencer. To generate tiled PCR amplicons from the SARS-CoV-2 viral cDNA, primers were designed using primal scheme. These primers were pooled into three different primer sets named as pool 1, 2 and 3.
Briefly, multiplex PCR was performed with initial denaturation at 98 °C for 30 s followed by 25 cycles of denaturation at 98 °C for 15 s and annealing and extension at 65 °C for 5 min. Amplification from respective primers sets were confirmed by agarose gel electrophoresis. Raw data generated was subjected to analysis using ARCTIC protocol to generate consensus sequences for SARS-CoV-2 genome.
Nanopore library preparation and sequencing
Amplified products obtained from multiplex PCR of pool 1, 2 and 3 were pooled and purified by using Ampure-XP beads. 50 ng amplified DNA from each samples was taken for library preparation using native barcoding kit and ligation kit from Oxford nanopore technology (ONT). Equimolar DNA from each sample was taken and end-repaired using NEBNext Ultra II End repair/dA-tailing module and cleaned with 0.4X Ampure XP Beads. Native barcode ligation was performed with NEBNext Ultra II Ligation module using Native Barcoding kit. All barcode ligated samples were pooled together and purified using 0.4X of AmPure beads. Further sequencing adapter ligation was performed using NEB next quick ligation module. Library mix was cleaned up using 0.4X AmPure beads and finally sequencing library was eluted in 15 μl of elution buffer and used for sequencing on SpotONflowcell in a 48 h sequencing protocol on GridION release 19.06.9. Nanopore raw reads (‘fast5’ format) were base called (‘fastq5’ format) and multiplexed using Guppy v3.2.2.
Data analysis
Base-calling and demultiplexing of nanopore raw data was done using Guppy. The processed data was used for variant calling using Arctic pipeline and consensus generation using bam based method. The Arctic pipeline uses processed reads to align against the available SARS-CoV-2 reference genome. Further, variant calling and consensus sequence generation was done using Nanopolish. The good quality variants were used for annotation using snpEff tool.
Genome analysis of SARS-CoV-2
The nucleotide sequences of representative SARS-CoV-2 were retrieved from Global Initiative on Sharing All Influenza Data (GISAID) and NCBI GenBank, edited and analysed employing EditSeq and MegAlign modules of lasergene5 software package (DNASTAR Inc, USA). The complete genome of 26 SARS-CoV-2 deciphered in this study was comparatively analysed with prototype SARS-CoV-2 isolated from Wuhan, China (GenBank Acc No MN908947). Multiple sequence alignment was carried out using MUSCLE alignment method in Bioedit software module. The amino acid substitutions of viruses sequenced in this study was compared to prototype SARS-CoV-2 Wuhan strain.
Phylogenetic analysis based on SARS-CoV-2 whole genomes were carried out with respect to globally diversified SARS-CoV-2 (n = 37) available at NCBI GenBank and GISAID data base from December 2019- May 2020 (Supplementary Table 2). The phylogenetic tree was constructed employing Neighbor Joining method with 1,000 replicates of bootstrap analysis with general time- reversible model with gamma distributed rates of variations among sites using Mega 5.03 software. Another S gene based phylogenetic tree was constructed by including 125 Indian SARS-CoV-2 strains from January 2020 to December 2020 employing the same Neighbor Joining method as described earlier.
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