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Researchers at the Botnar Research Centre have developed technology that facilitates standalone long-read Oxford Nanopore sequencing of single cells, opening avenues for understanding the causes of diseases like cancer.

During the development of cancer, changes occur at a molecular, cellular and tissue level. One of the aims of studying these changes is to identify features that can be co-opted as biomarkers for early cancer detection. Previously, researchers have mainly used bulk sequencing methods to investigate the molecular changes in a collection of cells. Single-cell genomics, the ability to examine all information contained in an individual cell, is a rapidly evolving field and can give a more detailed picture of the molecular alterations during cancer initiation.

“The application of accurate long-read single-cell sequencing will have a transformative effect on the wider single-cell sequencing community, as longer and full-length transcriptomic sequencing allows users to capture more information about the transcriptional and functional state of a cell,” says Assistant Professor Adam Cribbs, senior author of the paper and Group Leader in Systems Biology and Next Generation Sequencing Analysis at the Botnar Research Centre. “This means that we move closer to being able to better understand and diagnose diseases such as cancer”. 

Single-cell genomics is dominated by droplet-based short-read single-cell sequencing applications. In this approach, cells are encapsulated with barcoded RNA-capture microbeads into droplets within an oil emulsion. Each droplet becomes a discrete reaction vessel, associating a different barcode with each cell’s RNA and a unique molecular identifier (UMI) with each RNA transcript. Once barcoded, RNA from all cells can be pooled and processed conventionally for next-generation sequencing. During sequencing, both the original RNA sequence and the associated barcode and UMI are determined. Key to measuring abundance of each RNA and correctly associating them with their cell of origin is accurate assignment of the UMIs and barcodes. 

Long-read sequencing approaches, such as those of Oxford Nanopore Technologies, are currently revolutionising bulk sequencing approaches. “Long-read single-cell technology has the potential to interrogate not only RNA abundance, but also splice variants, structural variation and chimeric transcripts at the single-cell level. Collectively, the ability to determine these features accurately will improve diagnostics and biological understanding. However, Nanopore sequencing can be inaccurate, which hinders the critical steps of barcode and UMI assignment, making its application to single-cell sequencing challenging,” explains Dr Martin Philpott, first author of the paper and Director of the Next-Generation sequencing facility at the Botnar Research Centre.

To overcome these challenges, the team has developed a new approach called single-cell corrected long-read sequencing (scCOLOR-seq) that identifies and corrects errors in the barcode and UMI sequences, permitting standalone cDNA Nanopore sequencing of single cells. “Each mRNA molecule is tagged with a short sequence which identifies it within a certain droplet,” adds Dr Cribbs. “However, Nanopore long-read sequencing is too error prone to reliably sequence these tags, making it difficult to map the mRNA back to its specific cell. What we’ve been able to do is to develop a practical method for building redundancy into the tag, allowing inaccuracies within the sequencing to be pinpointed, and then correct them. The mRNA can then be linked back to an individual cell.”

“This study demonstrates an incredible cross-disciplinary team effort to advance single-cell technologies and is the result of strategic investments into these technologies at our department,” adds Professor Udo Oppermann, Director of Laboratory Sciences at the Botnar Research Centre and co-senior author of the paper. “We will continue our collaborative efforts to develop innovative single-cell approaches and - as demonstrated in the paper- apply this to molecular analyses in primary and secondary bone and other haematological cancers. Our intention is to advance these technologies in personalised medicine approaches such as cancer diagnosis allowing rational clinical decision making.” 

 

The work, in part supported by grants from the UKRI (Innovate UK, EPSRC and MRC), results from a collaboration with researchers from the Department of Chemistry at Oxford University, ATDBio, a world leader in complex oligonucleotide chemistry created by Professor Tom Brown at the Chemistry Department, University of Oxford, and pharmaceutical company Bristol Myers Squibbs. The study has been published in this week’s issue of Nature Biotechnology

 

Read the full story on the Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences website.