Rev Sel Coeff Mean

Growth/Fitness

Fitness

unitless

Illumina Sequencing , Deep Mutational Scanning , Viral Competition

None

None

None

None

None

None

Viral competition and selection: Each library, representing a randomized and unconstrained version of non-overlapped Tat (tat-in-nef) or Rev (rev-in-nef) at a single position, was competed against itself, raised in 293 cells, and then competed against itself in SupT1 cells (approx. 6 generations of selection) . Viral supernatants were harvested, genomic RNA isolated, and reverse transcribed. Amplicons targeting the randomized site were generated, indexed, normalized and then pooled and sequenced using PE150s on a MiSeq (Illumina). Selection experiments for each residue (202 total + reference wells) were performed in biological duplicate.

Data processing: Samples were de-multiplexed using MiSeqReporter to produce fastq files, each representing a single competition experiment. The resulting paired-end fastq files were then aligned to one another and the reference codon-swapped sequence (Table S2) and the frequencies of every codon were calculated. The randomized position was then extracted and a post-and pre-selection allele frequencies
calculated. Synonymous codons were pooled as correlations between amino acids across biological replicates was much stronger than individual correlations of codons (data not shown). See Methods for more details.

Relative fitness (Mean selection coefficient, from 2 replicates) was reported as a logarithmic ratio of the allele frequencies post- and pre-selection.