Targeting Long-read RNA Sequencing with Low-Cost Technology

Researchers at the Children’s Hospital of Philadelphia (CHOP) have created a flexible and affordable approach for targeted sequencing of full-length RNA molecules, which might hasten the development of novel diagnostics and therapies. When compared to commercially available targeted RNA sequencing methods, the technique, known as TEQUILA-seq, is remarkably cost-effective and can be used for a variety of scientific and medical applications. The specifics were covered in a study published in Nature Communications.

An RNA molecule can be cut and linked in a variety of ways on the path from gene to protein before being translated into a protein. By using a technique called alternative splicing, many proteins can be encoded by a single gene. Even though alternative splicing is involved in many biological processes, disorders in its regulation can result in harmful RNA molecules in conditions like cancer.

The number of RNA molecules (sometimes referred to as “transcript isoforms”) that are produced by a single gene must be precisely counted for researchers to comprehend how alternative splicing can lead to disease.

Utilizing “long-read” RNA sequencing systems, which sequence RNA molecules longer than 10,000 bases end-to-end and capture all of the transcript isoforms, is one technique to do this. However, the moderate sequencing yield of these long-read platforms has limited their general implementation, particularly in the clinical situation where it could be prohibitively costly to provide long-read RNA sequencing data at clinically meaningful depth.

Targeted sequencing, which includes enriching certain nucleic acid sequences of interest before sequencing, is a practical method that could significantly enhance the coverage of preset targets, but its widespread use has been hampered by the expense and complexity of target capture.

Targeted long-read RNA sequencing is a powerful strategy for elucidating the RNA repertoire for any predefined set of genes. However, existing technologies for targeted sequencing of full-length RNA molecules are either expensive or difficult to set up, putting them out of reach for many labs.”

Lan Lin, PhD, Study Co-Senior Author and Assistant Professor, Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia

Lin added, “TEQUILA-seq solves that problem by being both inexpensive and easy to use. The technology can be adapted by users for different purposes, and researchers can choose which genes they want to sequence and make the reagents for target capture in their own labs. This has the potential to accelerate discovery of new diagnostic and therapeutic solutions for a wide range of diseases.”

Hybridization capture-based enrichment, which employs small fragments of nucleic acids termed oligonucleotides as capture probes, is one technique that enables targeted sequencing. These oligonucleotides (often known as “oligos”) are biotin-tagged and created to hybridize to their targets based on complementary nucleic acid sequences, making it simple to capture and isolate the target sequences from a biological sample.

While commercially produced biotinylated capture probes are expensive and can only be employed for a small number of reactions, making the per-sample cost for each capture reaction high, hybridization capture-based enrichment is an effective technique for targeted sequencing.

Transcript Enrichment and Quantification Utilizing Isothermally Linear-Amplified Probes in Conjunction with Long-Read Sequencing, or TEQUILA-seq, was created by CHOP researchers to overcome this issue.

The nicking-endonuclease induced isothermal strand displacement amplification procedure, which can produce a high number of biotinylated capture probes from an inexpensive pool of non-biotinylated oligos as the templates, is a crucial advancement in TEQUILA-seq.

The researchers can produce 25 ug of TEQUILA probes, which can be utilized for at least 250 capture reactions, using just 2 ng of template oligos as an input. TEQUILA-seq is incredibly affordable and scalable for large target panels and numerous biological samples due to this novel approach for synthesizing capture probes.

The researchers used TEQUILA-seq for different gene panels using synthetic RNAs or human RNAs to measure the performance of the method. While being hundreds of times less expensive per capture response, TEQUILA probes outperformed commercial capture probes in target capture and enrichment. The researchers also showed that TEQUILA-seq could substantially enhance detection while maintaining target RNA molecule quantification.

Using cheap reagents and an easy experimental workflow, TEQUILA-seq allows us to deeply sequence the full-length RNA molecules for any gene set across many biological samples. This is very exciting and enables a wide range of medical applications, from RNA-guided genetic diagnosis to therapy development.”

Yi Xing, PhD, Director, Center for Computational and Genomic Medicine, Children’s Hospital of Philadelphia

The researchers used TEQUILA-seq to characterize full-length RNA molecules of 468 actionable cancer genes across 40 breast cancer cell lines to demonstrate its biological value. They found previously unidentified transcript isoforms in widely researched cancer genes that may provide insight into how cancer-fighting genes are turned off in certain malignancies.

Dr Lin added, “Our work shows that TEQUILA-seq can be broadly used for targeted sequencing of full-length RNA molecules. Moreover, TEQUILA probes are general-purpose capture probes. They are compatible for both targeted RNA and DNA sequencing, on both long-read and short-read sequencing platforms. The ability to easily generate large quantities of biotinylated capture probes for any target panel at a low cost and with ease can facilitate large-scale and population-level studies for many basic, translational, and clinical applications.”