Homologous pair of chromosome; Homozygous samples

1. Dissecting strand-bias dependent Misgenotyping Haplotype strands with greater thrmostable variant at SNP locus: Homozygote 3’ G G 3’ 5’ 5’ Homologous pair of chromosome; Homozygous samples

2. Thermodynamically greaterstable allele Homozygote Perfectly complimentary primer Steps 3’ C 5’ Perfect primer binding, G 3’ Template efficient polymerase binding Variable Site with greater thermo stable variant Taq Polymerase Fast polymerization reaction Insignificant number of template primer denaturation at extension temperature Efficient PCR for n cycles Very Large number of Amplicon Copy Large number of amplicon’s copy

3. Dissecting strand-bias dependent Misgenotyping Haplotype strands with less thrmostable variant at SNP locus: Other allele homozygote Homologous pair of chromosome; Homozygous samples 3’ T T 3’ 5’ 5’

4. Thermodynamically lessstable allele Homozygote Perfectly complimentary primer Steps 3’ A 5’ Perfect primer binding 3’ T Template efficient polymerase binding Variable Site with less thermo stable variant Taq Polymerase Fast polymerization reaction Insignificant number of template primer denaturation at extension temperature Efficient PCR for n cycles Similar very Large number of Amplicon Copy Similar large number of amplicon’s copy

5. Dissecting strand-bias dependent Misgenotyping Haplotype strands with two differentially thrmostable variant at SNP locus: heterozygote Homologous pair of chromosome; Homozygous samples 3’ G T 3’ 5’ 5’

6. Steps Perfectly complimentary primer Heterozygotetemplate with differentially thermostable haplotype strand (Due to the C/A SNP) Perfect primer binding to both strands 3’ C 5’ G 3’ efficient polymerase binding to both strand’s template primer duplex Template Variable Site with greater thermo stable variant Taq Polymerase Fast polymerization reaction for both of the strands Perfectly complimentary primer Insignificant number of template primer denaturation at extension temperature (both strand duplex) 3’ 5’ A T 3’ Efficient PCR for n cycles Template Variable Site with greater thermo stable variant Taq Polymerase Similar very Large number of amplicon copies of both of the strands, thus no amplification biasand thus no misgenotyping

7. Dissecting strand-bias dependent Misgenotyping Haplotype strands with greater thrmostable variant at SNP locus: Homozygote Site of mismatch; within primer binding site 3’ G 3’ G 5’ 5’ Homologous pair of DNA Fragments; Homozygous samples

8. Mismatch at 3rd position Thermodynamically greaterstable allele Homozygote Mismatch primer Steps 3’ C 5’ Imperfect & weaker primer binding G 3’ DNA Template polymerase binding partially hampered due to mismatch primer Variable Site with greater thermo stable variant Taq polymerase Slowerpolymerization reaction Good number of template primer denaturation at extension temperature PCR for n cycles Significantly less number of Amplicon Copy

9. Dissecting strand-bias dependent Misgenotyping Haplotype strands with less thrmostable variant at SNP locus: Other allele homozygote Site of mismatch; within primer binding site 3’ T Homologous pair of DNA Fragments; Homozygous samples 3’ T 5’ 5’

10. Mismatch at 3rd position Thermodynamically lessstable allele Homozygote Mismatch primer Steps 3’ A 5’ Imperfect & weaker primer binding T 3’ polymerase binding partially hampered due to mismatch primer DNA Template Variable Site with less thermo stable variant Taq polymerase Less thermostable variant make it Even more Slowerpolymerization reaction Even Greater number of template primer denaturation at extension temperature PCR for n cycles Even significantly less than the number of Amplicon Copies of previous weak amplification

11. Dissecting strand-bias dependent Misgenotyping Haplotype strands with two differentially thrmostable variant at SNP locus: heterozygote Site of mismatch; within primer binding site G 3’ 3’ T Homologous pair of DNA fragments; Heterozygous samples 5’ 5’

12. Heterozygotetemplate with differentially thermostable haplotype strand (Due to the C/A SNP) Imperfect & weaker primer binding with both the haplotype strand of heterozygote Greater thermostable strand Mismatch primer 3’ C 5’ G 3’ polymerase binding partially hampered due to mismatch primer for both of the strand equally DNA Template Variable Site with greater thermo stable variant Taq polymerase Differential slower polymerization reaction because of differential thermodynamic stability due to the immediate variable site Mismatch primer Less thermostable strand Differential number of template primer denaturation at extension temperature may occur due to differential thermodynamic stability 3’ A 5’ T 3’ DNA Template Variable Site with less thermo stable variant PCR for n cycles Taq polymerase Amplification Bias towards the more thermostable template strand (Strand Bias) Two homologous paired fragments of DNA (Heterozygote)

13. This strand bias will lead to misgenotyping. • During capillary sequencing, the chromatogram peaks for green variant carrying strand will be high. Chromatogram peak for the orange variant carrying strand will be very low (may seem like noise). Thus Heterozygote would be genotyped as homozygote. Sample No x, Homozygote Sample No x, Heterozygote