 Effective displayed-library coverage

Effective displayed-library coverage describes which molecular sequence variants actually occur as usable displayed molecules, rather than merely being allowed by a library design or present in its input material. In mRNA display, successful translation, fusion formation and recovery connect input sequences to usable genotype-phenotype linkages. Duplicates of one sequence do not replace missing sequences.

An original occupancy model has $S$ equally likely sequence variants, $N$ independent input molecules and usable-display probability $f$ for each input. A particular variant is usable with probability $f/S$ per input. Thereforewhere $D$ is the number of distinct represented variants. The expected usable molecule count is $Nf$, which is generally larger than the expected distinct count.

For $S=1000$, $N=300,000$ and stipulated $f=0.01$, the expected usable count is 3000 but the expected distinct count is only about 950.2. An average 95% represented fraction does not imply 95% probability that every variant is represented. By the union bound,A sufficient condition for that probability to be at most $\delta$ is $N\ge (S/f)\log (S/\delta )$. With $\delta =0.05$ in this example, this requires at least 990,349 input molecules under the stated model.

The figure uses a Poissonized displayed-library occupancy model: independently Poisson-distributed usable counts per variant with mean $\lambda =Nf/S$. The expected represented fraction is $1-e^{-\lambda }$, while the probability of all $S$ variants being present is $(1-e^{-\lambda }{)}^S$. That is an explicitly different count model, providing a convenient comparison rather than an exact fixed-input formula.

Real variants need not have equal abundance or display probability. In the original RNA-peptide fusion experiment, the selected and unselected encoding-sequence ratios were affected by preferential translation and reverse transcription.^[ref] Coverage calculations therefore need assumptions about sequence representation and process losses; nominal library size alone does not validate them.