Table 2.
Detection Methods for Low-Abundance Drug-Resistant HIV-1 Variants Used in the Included Publications
| Detection Method | Technical Strengths | Technical Limitations | Technical Cutoff Applied, % Min–Max |
|---|---|---|---|
| 454 pyrosequencing | Good sensitivity and specificity; long reads at short run times | Homopolymer errors; relatively high insertion/deletion rate; low throughput; costly reagents, (not available anymore) | 0.02–5 |
| AS-PCR | High sensitivity and specificity; fairly labor-intensive; easy interpretation of results | Only particular mutations of interest can be detected; false-positive results at lower limits; polymorphisms at primer binding sites can reduce assay’s sensitivity/specificity; varying sensitivity/specificity for different mutations due to virus- and assay- related issues | 0.001–2 |
| Cloning + sequencing | High sensitivity; not susceptible to primer polymorphisms; genetic linkage is possible if single genome amplification is applied | Time and labor intensive | 0.5–10; depending on sequenced clones |
| Illumina NGS | High-throughput data with low error rates; high sensitivity; relatively cheap | Fairly laborious with long run times | 1–3 |
| OLA | High sensitivity and specificity; fairly labor intensive; easy interpretation of results | Only particular mutations of interest can be detected; false-positive results at lower limits; polymorphisms can reduce sensitivity | 2 |
| RCA | High sensitivity and specificity; fairly labor intensive; easy interpretation of results | Only particular mutations of interest can be detected; false-positive results at lower limits; polymorphisms can reduce sensitivity | 1 |
| SGA | Risk of nucleotide misincorporation or template switching introduced during PCR amplification is reduced; genetic linkage is possible | Very labor intensive and costly; much time involved in determining the appropriate dilution to use | 2 |
| SMRTS (Pacific Biosciences) | Long reads; low error rate due to circular consensus sequencing | Fairly laborious; high input amount of DNA required | 1 |
Abbreviations: AS-PCR, allele-specific polymerase chain reaction; HIV, human immunodeficiency virus; NGS, next-generation sequencing; OLA, oligonucleotide ligation assay; RCA, rolling-circle amplification; SGA, single-genome amplification; SMRTS, single-molecule real-time sequencing.