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Determining the Precision of High-Throughput Sequencing and Its Influence on Aptamer Selection
Juan Canoura1, Obtin Alkhamis1, Caleb Byrd1
1Department of Chemistry, North Carolina State University, 2620 Yarbrough Dr., Raleigh, North Carolina 27695, United States.
Analytical Chemistry
|October 25, 2024
Summary
High-throughput sequencing (HTS) is crucial for in vitro selection, but its precision for measuring sequence abundance below 0.1% is unreliable. Accurate HTS data is vital for identifying functional nucleic acids and aptamers.
Area of Science:
- Molecular Biology
- Biotechnology
- Genomics
Background:
- In vitro selection identifies functional nucleic acids from randomized libraries.
- High-throughput sequencing (HTS) monitors oligonucleotide pool evolution during selection.
- The precision of HTS for aptamer selection is not well understood.
Purpose of the Study:
- To empirically determine the precision of HTS for measuring sequence abundance in in vitro selection.
- To assess the impact of HTS precision on aptamer discovery.
- To provide practical insights for researchers using HTS in nucleic acid selection.
Main Methods:
- Conducted multiple independent Illumina-based sequencing trials of in vitro selected pools.
- Empirically determined the precision of sequence abundance measurements.
- Analyzed HTS data reliability across different sequence abundance thresholds.
Main Results:
- HTS measurements for sequences with abundance ≥0.1% are generally reliable (RSD <25%).
- Measurements below the 0.1% abundance threshold are highly irreproducible.
- HTS data precision directly impacts the utility of enrichment metrics for predicting functional properties.
Conclusions:
- The reliability of HTS data is critical for successful in vitro selection and aptamer discovery.
- Practitioners must consider HTS precision limitations, especially for low-abundance sequences.
- These findings aid in optimizing HTS-based strategies for isolating and characterizing functional oligonucleotides.
