Determining the Precision of High-Throughput Sequencing and Its Influence on Aptamer Selection

<i>In vitro</i> selection offers a means of discovering functional nucleic acids from randomized libraries, and high-throughput sequencing (HTS) is a powerful tool for monitoring the evolution of oligonucleotide pools over many cycles of enrichment. Many groups now use HTS-derived measures of sequence enrichment across different rounds of <i>in vitro</i> selection to identify promising candidate sequences. However, the precision of HTS in this context─and its impact on the success or failure of the resulting aptamer selection process─remain poorly understood. Here, we conduct multiple independent Illumina-based sequencing trials of <i>in vitro</i> selected pools and empirically determine the precision of the resulting sequence abundance measurements. We find that measurements for sequences with abundance ≥0.1% are generally reliable, with relative standard deviations of <25%. Below this abundance threshold, however, such measurements are highly irreproducible. We demonstrate the practical utility of our findings with several case studies in which HTS data is used to accurately predict the functional properties of oligonucleotides enriched via <i>in vitro</i> selection. Our findings show that the utility of enrichment-based metrics is strongly dependent on the precision of the HTS data used to derive those metrics. These insights will prove beneficial for practitioners using HTS in isolating and characterizing functional oligonucleotides.