Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments
Abstract
:1. Introduction
2. Methods
2.1. Cell Sorting
2.2. Nucleotide Acid Purification and PCR Amplification
2.3. Sequencing and Phylogenetic Analyses
3. Results and Discussion
4. Conclusion
Author Contributions
Acknowledgments
Conflicts of Interest
References
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Sanger |
---|
First Round Forward (Protease):5′-TGAARGAITGYACTGARAGRCAGGCTAAT-3′ Reverse (Protease): 5′-AYCTIATYCCTGGTGTYTCATTRTT-3′ Forward (RT): 5′-TTTYAGRGARCTYAATAARAGAACTCA-3′ Reverse (RT): 5′-CCTCITTYTTGCATAYTTYCCTGTT-3′ |
Second Round Forward (Protease): 5′-YTCAGRCAGRCCRGARCCAACAGC-3′ Reverse (Protease): 5′-CTGGTGTYTCATTRTTKRTACTAGGT-3′ Forward (RT): 5′- TTYTGGGARGTYCARYTAGGRATACC-3′ Reverse (RT): 5′- GGYTCTTGRTAAATTTGRTATGTCCA-3′ |
NGS |
PR-INNER_F 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCTTTAACTTCCCTCAGGTCACTCT-3′ RT-1_R 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGTCAATGGCCATTGTTTAACTTTTGG-3′ RT-1_F 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCAAAAGTTAAACAATGGCCATTGAC-3′ PRNEWIN_R 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGCTGGTGTYTCATTRTTKRTACTAGGT-3′ 5FP127_F 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATACTGCATTTACCATACCTAG-3′ 3F262_R 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCCACTAACTTCTGTATGTC-3′ |
Samples | Protease Major Resistance Mutations | RT Major Resistance Mutations |
---|---|---|
267966 (Female) Viral load: 1450 copies/mL CD4+ T cell counts: 649 cells/µL Antiretroviral therapy: Yes | ||
Plasma Sanger | none | none |
Plasma NGS (consensus 20%) | none | M184V, E138EA |
PBMCs Sanger | none | M184V, E138A |
PBMC’s NGS (consensus 20%) | none | M184MV, E138EA |
CD4+ memory T-cells Sanger | none | M184V, E138A |
CD4+ memory T-cells NGS (consensus 20%) | none | M184V, E138A |
268138 (Male) Viral load: 12,500 copies/mL CD4+ T cell counts: 937 cells/µL Antiretroviral therapy: Not available | ||
Plasma Sanger | none | none |
Plasma NGS (consensus 20%) | none | none |
PBMCs Sanger | none | none |
PBMCs NGS (consensus 20%) | none | none |
CD4+ memory T-cells Sanger | none | none |
CD4+ memory T-cells NGS (consensus 20%) | none | none |
275163 (Female) Viral load: 16,105 copies/mL CD4+ T cell counts: 369 cells/µL Antiretroviral therapy: Yes | ||
Plasma Sanger | none | none |
Plasma NGS (consensus 20%) | none | none |
PBMCs Sanger | none | none |
PBMCs NGS (consensus 20%) | none | none |
CD4+ memory T-cells Sanger | none | none |
CD4+ memory T-cells NGS (consensus 20%) | none | none |
268249 (Female) Viral load: 32,468 copies/mL CD4+ T cell counts: 511 cells/µL Antiretroviral therapy: Yes | ||
Plasma Sanger | none | none |
Plasma NGS (consensus 20%) | none | none |
PBMCs Sanger | none | none |
PBMCs NGS (consensus 20%) | none | none |
CD4+ memory T-cells Sanger | none | none |
CD4+ memory T-cells NGS (consensus 20%) | none | none |
Sample | Gene | Classification | Surveillance | WT | Position | Mutation | Frequency | Coverage |
---|---|---|---|---|---|---|---|---|
267966 | ||||||||
Plasma | RT | NRTI | Yes | M | 184 | V | 99.4 | 46,490 |
RT | NNRTI | No | E | 138 | A | 99.29 | 55,090 | |
PBMCs | RT | NRTI | Yes | M | 184 | V | 42.23 | 69,897 |
RT | NNRTI | No | E | 138 | A | 42.29 | 69,891 | |
RT | Other | No | V | 179 | I | 41.47 | 69,896 | |
CD4+ memory T-cells | RT | NRTI | Yes | M | 184 | V | 99.12 | 54,457 |
RT | NNRTI | No | E | 138 | A | 99.38 | 54,452 | |
268249 | ||||||||
Plasma | PR/RT | - | - | WT | - | NP | - | - |
PBMCs | PR/RT | - | - | WT | - | NP | - | - |
CD4+ memory T-cells | PR | Other | No | L | 10 | V | 12.69 | 9382 |
268138 | ||||||||
Plasma | PR | Other | No | A | 71 | V | 99.17 | 20,609 |
PBMCs | PR | Other | No | A | 71 | V | 98.64 | 66,474 |
CD4+ memory T-cells | PR | Other | No | A | 71 | V | 98.49 | 79,020 |
275163 | ||||||||
Plasma | PR | Other | No | K | 20 | R | 23.13 | 49,598 |
PBMCs | PR | Other | No | K | 20 | R | 97.03 | 46,084 |
CD4+ memory T-cells | PR | Other | No | K | 20 | R | 96.9 | 33,222 |
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Arias, A.; López, P.; Sánchez, R.; Yamamura, Y.; Rivera-Amill, V. Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments. Int. J. Environ. Res. Public Health 2018, 15, 1697. https://doi.org/10.3390/ijerph15081697
Arias A, López P, Sánchez R, Yamamura Y, Rivera-Amill V. Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments. International Journal of Environmental Research and Public Health. 2018; 15(8):1697. https://doi.org/10.3390/ijerph15081697
Chicago/Turabian StyleArias, Andrea, Pablo López, Raphael Sánchez, Yasuhiro Yamamura, and Vanessa Rivera-Amill. 2018. "Sanger and Next Generation Sequencing Approaches to Evaluate HIV-1 Virus in Blood Compartments" International Journal of Environmental Research and Public Health 15, no. 8: 1697. https://doi.org/10.3390/ijerph15081697