Determining\nthe Precision of High-Throughput Sequencing\nand Its Influence on Aptamer Selection

<i>In vitro</i> selection offers a means of\ndiscovering\nfunctional nucleic acids from randomized libraries, and high-throughput\nsequencing (HTS) is a powerful tool for monitoring the evolution of\noligonucleotide pools over many cycles of enrichment. Many groups\nnow use HTS-derived measures of sequence enrichment across different\nrounds of <i>in vitro</i> selection to identify promising\ncandidate sequences. However, the precision of HTS in this contextand\nits impact on the success or failure of the resulting aptamer selection\nprocessremain poorly understood. Here, we conduct multiple\nindependent Illumina-based sequencing trials of <i>in vitro</i> selected pools and empirically determine the precision of the resulting\nsequence abundance measurements. We find that measurements for sequences\nwith abundance ≥0.1% are generally reliable, with relative\nstandard deviations of <25%. Below this abundance threshold, however,\nsuch measurements are highly irreproducible. We demonstrate the practical\nutility of our findings with several case studies in which HTS data\nis used to accurately predict the functional properties of oligonucleotides\nenriched via <i>in vitro</i> selection. Our findings show\nthat the utility of enrichment-based metrics is strongly dependent\non the precision of the HTS data used to derive those metrics. These\ninsights will prove beneficial for practitioners using HTS in isolating\nand characterizing functional oligonucleotides.