Table 1. Complete list of primary scores, Wald χ2, degrees of freedom and P values from logistic models for single-plex and 12-plex SNP-IT reaction.
Score name | Score description | Single-plex model χ2, df, P value | Multiplex model χ2, df, P value |
---|---|---|---|
TIN | The free energy of the most stable structure of extension primer with itself | 2.4, df = 1, P < 0.0001 | |
Most stable 3′-end dimer | The free energy of the most stable dimer formed by annealing SNP-IT primer with itself. Only the dimers formed by last nine 3′-end bases are considered for this score. In addition a binary becoming 1 when no dimers are formed at the 3′-end is used for single-plex model | 65.2, df = 1, P < 0.0001 | |
TDN | The free energy of the most stable structure between extension primer and PCR product | 28.1, df = 1, P < 0.0001 | 12.3, df = 1, P = 0.0005 |
Cumulative TDN | Free energies sum of all possible dimers (no loops) between extension primer and PCR product | 36.6, df = 1, P < 0.0001 | 7.1, df = 1, P = 0.0077 |
Number of consecutive Gs in SNP-IT primer | Binary score becomes 1 when number of consecutive Gs in SNP-IT primer is six and greater | 7.1, df = 1, P = 0.0078 | |
Stability of last 9 bases of extension primer | The free energy of the SNP-IT primer 3′-end. In addition to this score single-plex model uses its cube approximation and multiplex model uses its square approximation: | ||
Linear term of stability of last 9 bases of extension primer | 60.1, df = 1, P < 0.0001 | 284.1, df = 1, P < 0.0001 | |
Square term of stability of last 9 bases of extension primer | 172.0, df = 1, P < 0.0001 | ||
Cubic term of stability of last 9 bases of extension primer | 32.1, df = 1, P < 0.0001 | ||
Number of C3 linkers | Number of C3 linkers in the extension primer | 149.3, df = 1, P < 0.0001 | |
Binary score becomes one when number of C3 linkers is equal to 1 | 16.4, df = 1, P < 0.0001 | ||
Binary score becomes one when number of C3 linkers is greater than 3 | 8.9, df = 1, P = 0.0029 | ||
%GC of PCR primers | 29.7, d.f.=1, P < 0.0001 | 43.4, df = 1, P < 0.0001 | |
Amplicon melting temperature | 106.0, df = 1, P < 0.0001 | ||
Binary score becomes one when amplicon Tm is <73°C | 106.0, df = 1, P < 0.0001 | ||
Number of ambiguous bases | Number of ambiguous bases in amplicon | 27.6, df = 3, P < 0.0001 | |
Number of repeats | Number of repeats in amplicon | 109.1, df = 1, P < 0.0001 | |
Binary score becomes one when number of repeats in amplicon is 8 or 9 | 22.0, 1, P < 0.0001 | ||
Amplicon structure around SNP site | The free energy of the most stable amplicon structure (with loops) containing 5 bases of extension primer annealing site and 2 bases upstream of primer annealing site | 231.5, 1, P < 0.0001 | |
Amplicon structure around PCR primer annealing sites | The free energy of the most stable amplicon structure (with loops) containing 5 bases of PCR primer annealing site and 2 bases upstream of primer annealing site. Only one the most stable structure between two PCR primers was considered | 80.2, df = 1, P < 0.0001 | |
Extension mix change date and 13 interactions with combinations of extension mix and last 3′-end of SNP-IT | This binary score was introduced for the single-plex model to reflect historical assay modification. New extension nucleotides were introduced at Orchid Bioscience into SNP-IT assay | 491.6, df = 14, P < 0.0001a | |
Extension mix + last 2 bases at 3′-end of SNP-IT primer | 24 different combinations were used for the single-plex model and 67 combinations for the multiplex model out of 96 possible. Only combinations which show significant correlations with the failure/success rate were considered. In addition, 3 interactions scores with extension mix change date score are used for the single-plex model | 187.7, df = 24, P < 0.0001a | 543.0, df = 67, P < 0.0001a |
Extension mix + last 3 bases at 3′-end of SNP-IT primer | 42 different combinations were used for the single-plex model and 56 combinations for the multiplex model out of 384 possible. Only combinations which show significant correlations with the failure/success rate were considered. In addition, 7 interactions scores with extension mix change date score are used for the single-plex model | 208.2, df = 42, P < 0.0001a | 255.5, df = 56, P < 0.0001a |
Extension mix + last 4 bases at 3′-end of SNP-IT primer | 34 different combinations were used for the single-plex model and 60 combinations for the multiplex model out of 1536 possible. Only combinations which show significant correlations with the failure/success rate were considered | 101.8, p < 0.0001a | 155.1, p < 0.0001a |
Extension mix + last 5 bases at 3′-end of SNP-IT primer | 3 different combinations were used for the single-plex model and 18 combinations for the multiplex model out of 6144 possible. Only combinations which show significant correlations with the failure/success rate were considered | 9.3, P = 0.03a | 53.6, P < 0.0001a |
Last 2 bases at the 3′-end of the PCR primer + 1 amplicon base next to PCR primer annealing site | 2 different combinations were used for the single-plex model and 7 combinations for the multiplex model out of 64 possible. Only combinations which show significant correlations with the failure/success rate were considered | 5.9, P = 0.05a | 36.0, P < 0.0001a |
Last 3 bases at the 3′-end of the PCR primer + 1 base next to PCR primer annealing site | 22 different combinations were used for the single-plex model and 24 combinations for the multiplex model out of 256 possible. Only combinations which show significant correlations with the failure/success rate were considered | 91.1, P < 0.0001a | 70.5, P < 0.0001a |
Last 2 bases at the 3′-end of the PCR primer + 2 amplicon bases next to PCR primer annealing site | 19 different combinations were used for the single-plex model and 18 combinations for the multiplex model out of 256 possible. Only combinations which show significant correlations with the failure/success rate were considered | 67.4, P < 0.0001a | 48.5, P = 0.0001a |
Last 3 bases at the 3′-end of the PCR primer + 2 amplicon bases next to PCR primer annealing site | 40 different combinations were used for the single-plex model and 49 combinations for the multiplex model out of 1024 possible. Only combinations which show significant correlations with the failure/success rate were considered | 135.4, P < 0.0001a | 128.8, P < 0.0001a |
External TIN | The free energy of the most stable structure formed between marker extension primer and other extension primers in the multiplex cluster | 15.0, df = 1, P = 0.0001 | |
External TDN | The free energy of the most stable structure between marker extension primer and other PCR products in the multiplex cluster | 39.5, P < 0.0001 | |
External PCR TDN | The free energy of the most stable structure formed between any of two marker PCR primers and other PCR products in the multiplex cluster | 55.4, P < 0.0001 | |
Dispersion of amplicon melting temperature | The score is calculated as the sum of absolute differences between Tm of marker amplicon and Tm of all other amplicons in the multiplex cluster | 16.3, P < 0.0001 | |
Dispersion of amplicon complexity | The score is calculated as the sum of absolute differences between marker amplicon complexity and complexity of all other amplicons in the multiplex cluster | 22.6, P < 0.0001 |
An empty cell for the score indicates that it was not included into the corresponding model.
aThe difference in Wald χ2 between the final model and the model without the set of binary variables, degrees of freedom and P value.