Journal: PLoS Genetics
Article Title: Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality
Figure Lengend Snippet: Construction of the mutant libraries. (A) A schematic representation of the fitness profiling experiment is shown. A 240 bp insert was generated by error-prone PCR and BsaI digestion. The corresponding vector was generated by high-fidelity PCR and BsmBI digestion. Each of the nine plasmid libraries in this study consist of ∼ 50,000 clones. Each viral mutant library was rescued by transfecting ∼ 35 million 293T cells. Each infection was performed with ∼ 10 million A549 cells. (B) A schematic representation of the sequencing library preparation is shown. DNA plasmid mutant library or viral cDNA was used for PCR. This PCR amplified the 240 bp randomized region. The amplicon product was then digested with BpmI, end-repaired, dA-tailed, ligated to sequencing adapters, and sequenced using the Illumina MiSeq platform. BpmI digestion removed the primer region in the amplicon PCR, resulting in sequencing reads covering only the barcode for multiplex sequencing and the 240 bp region that was randomized in the mutant library. With this experimental design, the number of mutations carried by individual genomes in the mutant libraries could be precisely determined.
Article Snippet: The insert was then digested by BsaI (New England Biolabs, Ipswich, MA), whereas the vector was digested by BsmBI (New England Biolabs).
Techniques: Mutagenesis, Generated, Polymerase Chain Reaction, Plasmid Preparation, Clone Assay, Infection, Sequencing, Amplification, Multiplex Assay