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Table 3 In-house developed primers and RT-PCR protocol

From: Evaluation on the use of Nanopore sequencing for direct characterization of coronaviruses from respiratory specimens, and a study on emerging missense mutations in partial RdRP gene of SARS-CoV-2

Primer sequences
Forward 1 5′-ATG GGN TGG GAY TAY CC-3′ ~ 440 bp amplicon
Forward 2 5′-GGA YTA YCC NAA RTG YGA-3′
Reverse 5′-CCA TCA TCA SWN ARN ATS AT-3′
Reverse transcription
Master mix 1 constituents per reaction Temperature profile
Random hexamer 1 µL 65 °C
On ice
5 m
1 m
dNTP (10 mM) 1 µL
RNA template 8 µL
Total 10 µL
Master mix 2 constituents per reaction RT profile
10x RT buffer 2 µL   
MgCl2 (25 mM) 4 µL 25 °C 10 m
DTT (0.1 M) 2 µL 50 °C 50 m
RNaseOUT 1 µL 85 °C 5 m
SuperScript III reverse transcriptase 1 µL 15 °C Hold
Master mix 1 10 µL   
Total 20 µL   
PCR
Master mix constituents per reaction PCR profile
10x PCR buffer 5 µL    
MgCl2 (25 mM) 8 µL    
dNTP (10 mM) 1 µL 95 °C 9 m  
Forward primer 1 (200 µM) 1 µL 95 °C (1 °C/s) 1 m  
Forward primer 2 (200 µM) 1 µL 48 °C (1 °C/s) 1 m 40×
Reverse primer (200 µM) 1 µL 72 °C (1 °C/s) 1 m  
PCR-grade water 11.5 µL 72 °C 5 m  
AmpliTaq Gold 1.5 µL 15 °C Hold  
RT product 20 µL
Total 50 µL
  1. The primers were designed with reference to 56 coronavirus reference genomes
  2. Coronavirus reference genomes considered for designing primers
  3. NC_028752.1, NC_002645.1, NC_005831.2, NC_032107.1, NC_028824.1, NC_009988.1, NC_010437.1, NC_010438.1, NC_028814.1, NC_018871.1, NC_003436.1, NC_009657.1, NC_022103.1, NC_028811.1, NC_028833.1, NC_028806.1, NC_038861.1, NC_002306.3, NC_030292.1, NC_023760.1, NC_034972.1, NC_032730.1, NC_035191.1, NC_038294.1, NC_019843.3, NC_034440.1, NC_009020.1, NC_009019.1, NC_039207.1, NC_004718.3, NC_045512.2, NC_014470.1, NC_025217.1, NC_030886.1, NC_009021.1, NC_003045.1, NC_006213.1, NC_017083.1, NC_026011.1, NC_001846.1, AC_000192.1, NC_012936.1, NC_006577.2, NC_011547.1, NC_011549.1, NC_016993.1, NC_011550.1, NC_039208.1, NC_016992.1, NC_016991.1, NC_016996.1, NC_016994.1, NC_016995.1, NC_001451.1, NC_010800.1, NC_010646.1
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