Cross-contamination of commercial oligonucleotides with library-structured sequences.

Custom oligonucleotides (oligos) play a crucial role in molecular biology applications, including sequencing, polymerase chain reaction (PCR), and library construction. For highly sensitive techniques such as next-generation sequencing (NGS) library preparation, oligos must exhibit exceptional purity and accuracy. Here, we provide a perspective on the presence of unrelated, contaminating sequences in custom-made oligos using a case study of an RNA-protein crosslinked immunoprecipitation (CLIP-seq) experiment. These contaminants led to artefactual results in NGS data, rendering their results uninterpretable. Systematic investigation identified a contaminating human immunodeficiency virus (HIV) gag sequence embedded between Illumina-compatible adapter sequences, forming a functional 'library-structured' molecule capable of amplification and sequencing. Additionally, other nonspecific sequences, including fragments from unrelated oligos, were detected. By sharing our analysis strategy and outcomes, we aim to raise awareness within the research community and emphasize the need for more stringent quality controls in oligo synthesis and validation by commercial suppliers, safeguarding academic experimenters' time and resources. Furthermore, this perspective provides a systematic approach for the identification of the source of contaminating sequences in custom-made oligos designed for NGS library preparations.