Computational protocol: HIV-1 Genetic Diversity in Antenatal Cohort, Canada

Similar protocols

Protocol publication

[…] In cases in which viral load was >1,000 RNA copies/mL plasma, HIV-1 genotyping was performed by using a protocol (Virco BVBA, Mechelen, Belgium) based on sequencing of a 1,497-bp fragment of the HIV-1 pol gene (position 2253-3749). In cases in which viral load was <1,000 copies/mL, viral RNA was extracted from plasma, and a 524-bp pol segment (position 2597–3120) was amplified by using primers 3069R (5´-GGA TGG CCC AAA GGT TAA ACA-3´) and 3591F (5´-ATC CTA CAT ACA AAT CAT CCA T-3´) and the QIAamp 1-step reverse transcription–polymerase chain reaction (RT-PCR) method (Qiagen, Mississauga, Ontario, Canada). PCR conditions were 40 cycles consisting of 94°C for 30 s, 53°C for 1 min, and 72°C for 1 min, followed by extension at 72°C for 10 min. Amplicons were cloned into pPCR-Script (Stratagene, La Jolla, CA, USA) and sequenced by using dye terminator chemistry (Beckman-Coulter, Palo Alto, CA, USA).Sequences were aligned with references (2001) representing different HIV-1 subtypes (http://hiv-web.lanl.gov) () by using Clustal X version 1.81 (). Kimura 2-parameter distance matrices were assembled (transition/transversion ratio of 2) (,). Phylogenetic reconstructions were built according to the neighbor-joining method, and 1,000 bootstrap resamplings were performed to assess tree topology (MEGA version 2.1) (). Clade assessment was based on reliable grouping (>80% bootstrap) with reference sequences (). RIP version 1.9 (http://www.hiv.lanl.gov/content/hiv-db/RIPPER/rip_test.html) () was used to examine potential intersubtype recombinants, with gap stripping on, a window size of 200 characters, and a significance threshold of 90%. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Human immunodeficiency virus 2, Human immunodeficiency virus 1, Homo sapiens
Diseases HIV Infections