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Reflex: intramolecular barcoding of long-range PCR products for sequencing multiple pooled DNAs
James A Casbon1, Andrew F Slatter, Esther Musgrave-Brown
1Population Genetics Technologies Ltd., Babraham Institute, Babraham, Cambridgeshire CB22 3AT, UK.
Nucleic Acids Research
|April 13, 2013
Summary
We developed Reflex™, a novel intramolecular reaction, to create shorter, sequencer-ready DNA products from pooled long-range PCR samples. This method efficiently preserves DNA barcodes, enabling cost-effective, uniform sequencing coverage for large-scale projects.
Area of Science:
- Molecular Biology
- Genomics
- Biotechnology
Background:
- Long-range polymerase chain reaction (PCR) generates large DNA fragments that can be challenging for next-generation sequencing (NGS).
- Achieving uniform sequence coverage across long contiguous targets in numerous samples is often costly and complex.
- Preserving unique DNA barcodes during sample processing is crucial for accurate multiplexed sequencing.
Purpose of the Study:
- To introduce Reflex™, an intramolecular reaction for processing pooled barcoded long-range PCR products.
- To enable the generation of shorter, sequencer-ready DNA fragments.
- To facilitate cost-effective and uniform sequence coverage of long DNA targets in high-throughput applications.
Main Methods:
- Development of the Reflex™ intramolecular reaction.
- Application of a minimal number of primer extension steps.
- Pooling of barcoded long-range PCR products.
- Sequencing on desktop next-generation sequencers.
Main Results:
- Successful derivation of shorter, sequencer-ready daughter products from long-range PCR amplicons.
- Preservation of cognate DNA barcodes throughout the Reflex™ workflow.
- Demonstration of uniform sequence coverage across long contiguous targets.
- Enabling of high-throughput analysis at low cost.
Conclusions:
- The Reflex™ workflow offers an efficient and cost-effective solution for preparing long-range PCR products for NGS.
- This method simplifies the process of achieving uniform sequence coverage for large-scale genomic studies.
- Reflex™ is suitable for low-cost, high-throughput sequencing applications using desktop NGS platforms.
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