Table 3 Comparison of individual allele frequencies from different mixtures of BH10 WT and Mutant RNA transcripts analyzed by all methods
From: Ultrasensitive single-genome sequencing: accurate, targeted, next generation sequencing of HIV-1 RNA
Method | qPCR cDNA input copies | Consensus sequences | Allele frequency expected (%) | % allele frequency detected | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
65R | 67N | 70R | 74V | 100I | 103N | 181C | 184V | 188C | 190A | ||||
a. uSGS vs Long Primer PCR-1 at 50% majority cutoff | |||||||||||||
uSGS | 179,825 | 41,050 | 10 | 15.2 | 15.7 | 15.7 | 15.7 | 15.8 | 15.8 | 16.0 | 16.0 | 16.0 | 16.0 |
LP-PCR-1 | 179,825 | 32,492 | 10 | 1.90 | 2.00 | 2.03 | 1.07 | 2.80 | 2.94 | 7.04 | 7.06 | 7.07 | 7.08 |
uSGS | 178,175 | 28,061 | 1 | 1.53 | 1.60 | 1.58 | 1.59 | 1.62 | 1.62 | 1.65 | 1.65 | 1.65 | 1.64 |
LP-PCR-1 | 178,175 | 20,565 | 1 | 0.22 | 0.23 | 0.24 | 0.12 | 0.31 | 0.32 | 0.69 | 0.70 | 0.70 | 0.70 |
b. uSGS vs Long Primer PCR-1 at 80% majority cutoff | |||||||||||||
uSGS | 179,825 | 41,050 | 10 | 8.2 | 8.5 | 8.5 | 8.5 | 8.5 | 8.5 | 8.5 | 8.5 | 8.5 | 8.5 |
LP-PCR-1 | 179,825 | 32,492 | 10 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.07 | 0.27 | 0.27 | 0.27 | 0.27 |
uSGS | 178,175 | 28,061 | 1 | 0.39 | 0.40 | 0.39 | 0.40 | 0.40 | 0.40 | 0.40 | 0.40 | 0.41 | 0.41 |
LP-PCR-1 | 178,175 | 20,565 | 1 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.02 | 0.04 | 0.05 | 0.04 | 0.04 |
c. uSGS vs Long Primer PCR-1 without Primer ID consensus builds | |||||||||||||
uSGS | 179,825 | 41,050 | 10 | 18 | 18.8 | 18.9 | 18.8 | 18.5 | 18.75 | 19 | 19.5 | 19.5 | 19.4 |
LP-PCR-1 | 179,825 | 32,492 | 10 | 6.8 | 8.5 | 8.4 | 6.9 | 8.6 | 8.8 | 9.2 | 9.3 | 9.0 | 8.8 |
uSGS | 178,175 | 28,061 | 1 | 1.8 | 2.0 | 1.9 | 1.9 | 1.9 | 1.9 | 2.1 | 2.1 | 2.1 | 2.1 |
LP-PCR-1 | 178,175 | 20,565 | 1 | 0.83 | 0.74 | 0.83 | 0.58 | 0.78 | 0.8 | 0.96 | 1.3 | 0.86 | 0.86 |
d. uSGS vs Long Primer PCR-2 and Long Primer PCR-3 at 50% majority cutoff | |||||||||||||
uSGS | 69,000 | 23,048 | 30 | 30 | 31 | 32 | 31 | 32 | 32 | 32 | 32 | 32 | 32 |
LP-PCR-2 | 69,000 | 17,230 | 30 | 21 | 22 | 22 | 22 | 22 | 23 | 23 | 23 | 23 | 23 |
LP-PCR-3 | 69,000 | 14,229 | 30 | 23 | 24 | 24 | 24 | 25 | 26 | 28 | 28 | 28 | 28 |
uSGS | 74,800 | 17,270 | 3 | 4.7 | 4.9 | 4.9 | 4.9 | 4.9 | 4.9 | 4.9 | 4.9 | 4.9 | 4.9 |
LP-PCR-2 | 74,800 | 14,451 | 3 | 3.1 | 3.2 | 3.3 | 3.3 | 3.3 | 3.3 | 3.3 | 3.3 | 3.3 | 3.3 |
LP-PCR-3 | 74,800 | 10,915 | 3 | 0.6 | 0.7 | 0.7 | 0.8 | 2.2 | 2.6 | 4.2 | 4.2 | 4.2 | 4.2 |
uSGS | 67,860 | 16,757 | 0.3 | 0.35 | 0.38 | 0.36 | 0.36 | 0.36 | 0.37 | 0.37 | 0.37 | 0.36 | 0.36 |
LP-PCR-2 | 67,860 | 15,696 | 0.3 | 0.32 | 0.30 | 0.33 | 0.31 | 0.32 | 0.36 | 0.32 | 0.32 | 0.32 | 0.32 |
LP-PCR-3 | 67,860 | 9546 | 0.3 | 0.02 | 0.02 | 0.02 | 0.03 | 0.02 | 0.10 | 0.31 | 0.31 | 0.32 | 0.32 |
e. uSGS vs Long Primer PCR-2 and Long Primer PCR-3 at 80% majority cutoff | |||||||||||||
uSGS | 69,000 | 23,048 | 30 | 29 | 30 | 30 | 30 | 30 | 30 | 30 | 31 | 31 | 31 |
LP-PCR-2 | 69,000 | 17,230 | 30 | 12 | 12 | 12 | 13 | 17 | 17 | 21 | 22 | 22 | 22 |
LP-PCR-3 | 69,000 | 14,229 | 30 | 1.8 | 1.9 | 2.1 | 2.1 | 4.4 | 5.3 | 24 | 25 | 26 | 26 |
uSGS | 74,800 | 17,270 | 3 | 4.3 | 4.5 | 4.5 | 4.5 | 4.6 | 4.6 | 4.6 | 4.9 | 4.9 | 4.9 |
LP-PCR-2 | 74,800 | 14,451 | 3 | 1.1 | 1.1 | 1.2 | 1.2 | 2.0 | 2.0 | 3.2 | 3.3 | 3.3 | 3.3 |
LP-PCR-3 | 74,800 | 10,915 | 3 | <0.01 | <0.01 | 0.01 | 0.01 | 0.02 | 0.02 | 2.6 | 3.0 | 3.2 | 3.2 |
uSGS | 67,860 | 16,757 | 0.3 | 0.28 | 0.31 | 0.29 | 0.29 | 0.32 | 0.32 | 0.33 | 0.36 | 0.36 | 0.36 |
LP-PCR-2 | 67,860 | 15,696 | 0.3 | 0.11 | 0.10 | 0.10 | 0.11 | 0.21 | 0.22 | 0.30 | 0.32 | 0.31 | 0.32 |
LP-PCR-3 | 67,860 | 9546 | 0.3 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | <0.01 | 0.12 | 0.12 | 0.17 | 0.19 |