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[…] Total cellular DNA was isolated from each p24-negative culture well using a QIAamp DNA midi kit (Qiagen) and screened for the presence of HIV provirus by nested PCR using primers specific to the Gag or RT genes (Integrated DNA Technologies). A 9kb, near-full length product was amplified in limiting dilution format from wells screening positive for non-induced provirus, as previously described [,]. The Gag gene was amplified from the near-full length amplicon, bead purified (Agencourt AMPure XP), and sequenced by Sanger methods on the Applied Biosystems 3730xl DNA Analyzer in the Genetic Resources Core Facility (Johns Hopkins School of Medicine, Baltimore, MD). Gag sequences were aligned to the HXB-2 reference sequence (CodonCode Aligner v4.04) and inspected for deletions. APOBEC3G-mediated G-to-A hypermutation was assessed using the Los Alamos National Laboratory Hypermut Tool (http://www.hiv.lanl.gov/content/sequence/HYPERMUT/hypermut.html).Four overlapping, nested PCR reactions were prepared from the 9kB, near-full length amplicons that demonstrated intact Gag sequences and purified, sequenced, and analyzed as above. Sequences were categorized as “Not Evaluable” when the 9kb template was depleted prior to obtaining near-full length sequence. Deletions with undefined junctions were defined by the identification of human sequence interspersed among HIV sequence or failure of at least three sequencing primers and PCR amplicons with smaller than expected size upon gel electrophoresis. The sensitivity of these methods was evaluated by amplification and sequencing of serial dilutions of the HIV-1 strain, BaL (data not shown). Additionally, full-length replication-competent sequences were detected from p24+ QVOA culture wells (data not shown).Sequences were aligned to the HXB2 reference genome using the MAFTT algorithm in Geneious software (v9.1). The initial alignment was proofed and edited by hand to ensure proper placement of nucleotides in gapped/truncated columns. A neighbor-joining tree was produced for specific regions of genome with high coverage in MEGA (6.0) using the Tajima-Nei molecular model, 1000 bootstrap replicate samples for statistical support, and rooted using the HXB-2 reference sequence. Trees were viewed and colored in Figtree software v1.4 (http://tree.bio.ed.ac.uk/software/figtree). […]

Pipeline specifications

Software tools CodonCode Aligner, Geneious, FigTree
Organisms Human immunodeficiency virus 1, Homo sapiens
Diseases Infection, Wiskott-Aldrich Syndrome, HIV Infections, Genomic Instability