Cross-contamination of commercial oligonucleotides with library-structured sequences

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Custom oligonucleotides (oligos) are vital for molecular biology techniques like sequencing and PCR.
• High purity and accuracy of oligos are critical for sensitive applications such as next-generation sequencing (NGS) library preparation.

Purpose of the Study:

• To investigate the impact of contaminating sequences in custom oligos on NGS experiments.
• To present a case study of artefactual results in an RNA-protein crosslinked immunoprecipitation sequencing (CLIP-seq) experiment due to oligo contaminants.
• To provide a systematic approach for identifying the source of contaminating sequences in custom oligos for NGS.

Main Methods:

• Case study analysis of a CLIP-seq experiment with artefactual NGS data.
• Systematic investigation to identify contaminating sequences within custom-made oligos.
• Bioinformatic analysis to detect unexpected sequences, including adapter-ligated fragments.

Main Results:

• Contaminating sequences, including a human immunodeficiency virus (HIV) gag sequence, were identified within custom oligos.
• These contaminants formed functional 'library-structured' molecules capable of amplification and sequencing.
• Artefactual NGS data rendered the experimental results uninterpretable, highlighting issues with oligo purity.

Conclusions:

• Contaminating sequences in custom oligos can lead to significant experimental errors and uninterpretable data.
• There is a critical need for enhanced quality control and validation processes by commercial oligo synthesis suppliers.
• Sharing analysis strategies can raise community awareness and promote better practices in oligo procurement for NGS applications.