Digital Quantification of Chemical Oligonucleotide Synthesis Errors.

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Chemically synthesized oligonucleotides are essential for nucleic acid technologies.
• Oligonucleotide sequence errors pose challenges for sensitive applications.
• Accurate identification and quantification of errors in synthetic oligonucleotides are difficult.

Purpose of the Study:

• To quantify errors in chemically synthesized oligonucleotides from various sources.
• To investigate the impact of synthesis strategies, purity, batches, and sequence context on oligonucleotide errors.

Main Methods:

• Utilized a digital sequencing approach with unique molecular identifiers.
• Analyzed oligonucleotides from multiple manufacturers with diverse synthesis methods and purity grades.
• Examined error types (deletions, substitutions) and their frequencies.

Main Results:

• Deletions were the predominant error type, occurring 7 times more frequently than substitutions.
• 97.2% of analyzed oligonucleotide molecules were intact, with deletions ranging from 0.2% to 11.7%.
• Significant batch-to-batch variability was observed, with batch effects potentially outweighing purification impacts. Deletion rates showed a 5'-end bias in certain sequence contexts. Oligonucleotide quality directly affects sequencing assay performance.

Conclusions:

• Oligonucleotide errors are influenced by manufacturer, synthesis strategy, purity, batch, and sequence context.
• Careful consideration of these factors is necessary when selecting and evaluating oligonucleotides.
• High-quality oligonucleotides are critical for reliable performance in molecular applications, including clinical diagnostics.