Positive ~9kb single genome amplicons were gel-extracted using the Wizard SV Gel and PCR Clean-Up System (Promega). Purified ~9 kb PCR amplicons were sent for sequencing to the University of Alabama Birmingham (UAB) sequencing core for Sanger sequencing.
In conjunction, multiple amplicons from recipient 3576 were sequenced by single-molecule nucleic acid sequencing (Pacific Biosciences), to confirm the TF [49 (link)]. Briefly, SMRTbell libraries were constructed according to the manufacturer's instructions for 10kb amplicons. PCR reactions of DNA amplicons were purified using Wizard SV Gel and PCR Clean-Up System (Promega) and mixed at equal concentrations to a total of 3ug DNA. Library preparation quality was assessed on a Bioanalyzer and SMRT sequencing on the PacBio RSII was performed following primer annealing and P4 polymerase binding to the library preparations. The consensus of the reads, aligned to the HXB2 reference sequence, were then taken to form a TF sequence, which matched the Sanger sequence.
In conjunction, multiple amplicons from recipient 3576 were sequenced by single-molecule nucleic acid sequencing (Pacific Biosciences), to confirm the TF [49 (link)]. Briefly, SMRTbell libraries were constructed according to the manufacturer's instructions for 10kb amplicons. PCR reactions of DNA amplicons were purified using Wizard SV Gel and PCR Clean-Up System (Promega) and mixed at equal concentrations to a total of 3ug DNA. Library preparation quality was assessed on a Bioanalyzer and SMRT sequencing on the PacBio RSII was performed following primer annealing and P4 polymerase binding to the library preparations. The consensus of the reads, aligned to the HXB2 reference sequence, were then taken to form a TF sequence, which matched the Sanger sequence.
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