Identifying sequencing artifacts in low-diversity, short-insert aptamer libraries on the Illumina MiSeq platform

Next-generation sequencing (NGS) of DNA aptamer screening libraries on the popular Illumina MiSeq platform revealed unexpected artifacts affecting data quality and sequencing efficiency. These 80-base long library sequences formed adapter-ligated side products, complicating size-based purification for separating the desired adapter ligation product from the undesired side product. Among these side products, low-input samples exhibited disproportionately high adapter trimer formation (40-50% vs. ∼1% in other samples) which resulted in overestimation of sample concentration and, in turn, decreased sequencing yield. Sequencing runs displayed highly variable pass rates (6-93%), likely due to short sequences outcompeting longer ones and assignment errors of neighboring clusters with identical early-cycle bases. Unexpected duplicates in template samples suggest that adapter-ligated daughter strands and optical misidentifications contribute to artificial duplication. These duplication artifacts are of particular importance in amplicon sequencing for which copy-number analysis is crucial. These findings highlight the need for refined ligation conditions, improved purification strategies, and enhanced bioinformatic filtering to ensure sequencing accuracy in aptamer screening library workflows on the Illumina platform.