This article reports on a study that used multiple independent Illumina-based sequencing trials of in vitro selected pools, in order to empirically determine the precision of the resulting sequence abundance measurements, noting findings that indicate that measurements for sequences with abundance ≥0.1 percent are generally reliable.
In vitro 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 in vitro 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, the authors conduct multiple independent Illumina-based sequencing trials of in vitro selected pools and empirically determine the precision of the resulting sequence abundance measurements. They find that measurements for sequences with abundance ≥0.1 percent are generally reliable, with relative standard deviations of <25 percent. Below this abundance threshold, however, such measurements are highly irreproducible. The authors 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 in vitro selection. Their 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. (Published Abstract Provided)
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