Sample collection and cDNA preparation
Nasal and oral samples were collected in the health care facilities in the south-west part of Bangladesh and sent to the genome center, Jashore University of Science and Technology, Bangladesh. RNA was extracted from those samples using nucleic acid extraction kit, Invitrogen inc. The extracted RNA was then tested for SARS CoV-2 using a commercial kit from Sansure Biotceh Co., Ltd (China). The left-over RNA were preserved at -40°C in the genome center lab. A total of 24 randomly selected SARS CoV-2 positive samples were tested for the analysis (supplementary Table s1 ). A representative SARS CoV-2 negative sample (of 5 of the negative samples randomly selected) was used as a negative control in this study (supplementary Table s2 ). cDNA was prepared for each selected sample using the GoScript™ Reverse Transcription System (Promega, USA) following the manufacturer’s protocol. In brief, primer/ RNA mix was prepared by mixing 10µl of extracted RNA with 1µl of Random primer and 1µl of Oligo(dT)15 primer (total volume 12µl). Then the mixture was heated at 70°C for 5 minutes, followed by immediate chilling on ice for 5 minutes and a quick spin. The mixture for reverse transcription reaction was prepared by making a cocktail of the components from GoScript™ Reverse Transcription System in a sterile 1.5ml micro centrifuge tube keeping on ice. The final reaction mix was 40μl for each cDNA synthesis reaction to be performed.