Sequencing Illumina libraries at high accuracy on the ONT MinION using R2C2

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• High-throughput short-read sequencing, predominantly using Illumina technology, is crucial for research and diagnostics.
• The high capital cost of Illumina sequencers limits adoption of alternative short-read technologies.
• Oxford Nanopore Technologies (ONT) offers low-cost, long-read sequencing with the MinION, but its error-prone nature can be a limitation.

Purpose of the Study:

• To adapt existing short-read Illumina libraries for use with the ONT MinION sequencer.
• To generate high-accuracy consensus reads from short DNA fragments using long-read sequencing technology.
• To demonstrate the versatility of this approach across various sequencing library types and applications.

Main Methods:

• Utilized the rolling circle to concatemeric consensus (R2C2) method to circularize and amplify short DNA library molecules.
• Generated longer DNA molecules containing tandem repeats of the original short library fragments.
• Sequenced the amplified DNA on the ONT MinION and processed reads to create high-accuracy consensus sequences.

Main Results:

• Successfully generated long DNA molecules suitable for ONT MinION sequencing from short Illumina libraries.
• Achieved high-accuracy consensus reads comparable to Illumina MiSeq error rates.
• Demonstrated and benchmarked the method using RNA-seq, ChIP-seq, and Tn5 libraries (regular and target-enriched).
• Implemented a real-time analysis workflow for rapid evaluation of sequencing library metrics.

Conclusions:

• The R2C2 method enables the use of low-cost ONT MinION sequencing for applications typically reliant on Illumina short-read technology.
• This approach significantly enhances the accessibility of high-quality sequencing data by leveraging existing library preparation methods with affordable hardware.
• The developed workflow offers a cost-effective and efficient alternative for various sequencing applications and library quality control.