Supplementary Table S1: Improved SARS-COV-2 PCR detection and genotyping with double-bubble primers

Table S1 Primers assignment. GAPDH- Glyceraldehyde 3-Phosphate Dehydrogenase. “N”- SARS-COV2 nucleocapsid gene. “S”- Spike gene. Normal Type primer - conventional primer usually 20bp. D-B Type primer- Double-Bubble primer with stem-loop and homo-dimer configurations. TqMTaqMan. Sense- 5’-->3’ primer orientation, AS- anti- sense 3’à5’ primer orientation. Ntnucleotide numbers in primer. Amplicon- size of PCR product in base pairs (bp). Region 1: primers # 3-8 in SARS-COV-2 gene “N”. Region 2: primers #9 and #10 in SARS-COV-2 gene “N”. Region 3: primer #11 in SARS-COV-2 gene “S”. We also designed 2 TaqMan (TqM) probes with non-fluorescent quencher (NFQ) and 2 different reporter dyes for each region (see also Fig. S3; probes #12 and #13, 45nt and 44nt, VIC and FAM dyes, regions 1 and 2, No Gene Type Orient Nt Sequence Amplicon (bp) Region 1 GAPDH Normal Sense 20 GGAAGGTGAAGGTCGGAGTC 135 1’ GAPDH Normal AS 25 ACATGTAAACCATGTAGTTGAGGTC 135 2 GAPDH D-B Sense 30 cacctatggg GGAAGGTGAAGGTCGGAGTC 152 2’ GAPDH D-B AS 32 atggttaACATGTAAACCATGTAGTTGAGGTC 152 3 “N” Normal Sense 21 GCAGAGACAGAAGAAACAGCA 96 1 3’ “N” Normal AS 20 TCAGCACTGCTCATGGATTG 96 1 4 “N” D-B Sense 30 ctgtct gacGCAGAGACAGAAGAAACAGCA 115 1 4’ “N” D-B AS 30 gcagtgactgTCAGCACTGCTCATGGATTG 115 1 5 “N” Normal Sense 20 CAAGCCTTACCGCAGAGACA 119 1 5’ “N” Normal AS 20 GCCTGAGTTGAGTCAGCACT 119 1 6 “N” Normal Sense 30 TGATGAAACTCAAGCCTTACCGCAGAGACA 139 1 6’ “N” Normal AS 30 ATGAGTTTAGGCCTGAGTTGAGTCAGCACT 139 1 7 “N” Normal Sense 30 ataaccaccaCAAGCCTTACCGCAGAGACA 139 1 7’ “N” Normal AS 30 atatttgactGCCTGAGTTGAGTCAGCACT 139 1 8 “N” D-B Sense 30 taaggccaaaCAAGCCTTACCGCAGAGACA 139 1 8’ “N” D-B AS 30 aactcagctcGCCTGAGTTGAGTCAGCACT 139 1 9 “N” Normal Sense 20 TCTTGCTTTGCTGCTGCTTG 138 2 9’ “N” Normal AS 20 GCAGTACGTTTTTGCCGAGG 138 2 10 “N” D-B Sense 30 aagcaatgggTCTTGCTTTGCTGCTGCTTG 158 2 10’ “N” D-B AS 30 cgtacgacgaGCAGTACGTTTTTGCCGAGG 158 2 11a “S” D-B Sense 31 aaaccacggcgATATGGTTTCCAACCtACTa 111 3 11b “S” D-B Sense 30 aaaccacgcgATATGGTTTCCAACCtACTt 111 3 11c “S” D-B AS 30 gtggacgacgTAGGTCCACAAACAGTTGCT 111 3 12 “N” TqM Sense 45 VIC-ACTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTG -NFQ-MGB 1 13 “N” TqM Sense 44 FAM-CAAGGCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAA -NFQ-MGB 2 respectively). The TqM probes were longer than the normal TqM probes to afford binding at higher annealing temperatures to take advantage of the longer D-B primers, for improved specificity and shorter PCR cycling. However, the TqM probe could not exceed the 45nt length since at longer molecular distance, the NFQ quenching capabilities of the reporter dye are not effective. In addition, the TqM probes included a minor groove binder (MGB) moiety at the 3’- end that increases the melting temperature (Tm) of the probe and stabilizes probe/target hybrids (ThermoFisher Scientific). Regions in D-B primers: Uppercase letters- sequence-specific. Uppercase black letters- 3’-overhang. Uppercase red bold letters and lowercase red letters- stem region. Uppercase red letters and lowercase green letters- loop region. Lowercase blue background letters in 3’-end of primer 13a: wild type “a”, and primer 13b- mutant “t”. Lowercase blue letter “t” in position minus 5 in primers 11a and 11b represents mismatch càt mutation introduced to weaken the primer annealing to its target. Locations of primers in the SARS-COV-2 genome are shown in Figure S3. Primer analysis was performed using IDT’s Oligo Analyzer Version 3.1. Care was taken to avoid primer-hetero-dimers.