fitness (log-fold expansion of each mutant relative to the population)

Growth/Fitness

Fitness

unitless

Illumina Sequencing , Deep Mutational Scanning , OD

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Plasmid DNA was extracted from frozen cell samples (Qiagen) and used as a template for PCR reactions (20 cycles) with custom barcoded primers containing Illumina flowcell adaptor sequences. The samples were multiplexed and run on an Illumina HiSeq instrument. Multiplexed Illumina reads from a single lane were sorted based on an exact match to a four-letter barcode sequence. Reads were then filtered to remove sequences that (a) contained frameshift mutations, (b) encoded for a parD3 variant not in the planned library, or (c) lacked an exact match to six nucleotides before (AGGCAG) and after (GCAAGC) the randomized region. Sequences that passed these quality filters were then counted and frequency-normalized. We calculated the fitness of each variant as described previously (van Opijnen et al., 2009). Briefly, we generated a linear fit to the frequencies of each mutant as a function of time, and then calculated the log-fold expansion of each mutant relative to the rest of the population, yielding Wraw for each variant: where t0 is the frequency of the mutant at 200 min, t1 is the frequency of the mutant at 600 min, and E is the expansion factor of the culture (OD at t0 / OD at t1).We then transformed these raw fitness values such that the W value for frameshift variants was 0 and the W value for the wild-type (LWDK) sequence was 1.