Identification of an abundant contaminant in seCLIP libraries

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 (NGS) library preparation, oligos must exhibit exceptional purity and specificity. Here, we report the unexpected presence of cross-contaminating sequences in commercially synthesized, PAGE-purified DNA oligos ordered for RNA-protein crosslinked immunoprecipitation (CLIP-seq) experiments. These contaminants led to significant artefacts in sequencing data, rendering results uninterpretable. Systematic investigation identified a contaminating HIV gag sequence embedded between Illumina-compatible adapter sequences, forming a functional library molecule capable of amplification and sequencing. Additionally, other non-specific sequences, including fragments from unrelated oligos, were detected within the supplied oligos. By sharing this discovery, we aim to raise awareness within the research community and emphasize the need for stringent quality controls in oligonucleotide synthesis and validation. Furthermore, this study provides a systematic method for identifying hard-to-detect contaminating sequences in commercial oligonucleotides designed for NGS library preparations.