Researchers have developed a new approach, called single-cell corrected long-read sequencing (scCOLOUR-seq), that is able to amend errors in long-read sequencing methods.
The rapidly evolving field of single-cell genomics is dominated by droplet-based short-read single-cell sequencing applications. This approach involves individual cells being isolated and contained in droplets with barcoded RNA-capture beads. Each droplet then becomes a reaction vessel – a different barcode is associated with each cell’s RNA and a unique molecular identifier (UMI) is linked to each RNA transcript. UMIs are short sequences that provide error correction and increased accuracy during sequencing by uniquely tagging each molecule in a sample library.
The RNA from all cells is then processed for next generation sequencing (NGS). Accurate assignment of the UMIs and barcodes is essential for measuring the abundance of each RNA in their cell of origin.
Single-cell corrected long-read sequencing (scCOLOR-seq)
Examples of long-read sequencing approaches are PacBio single-molecule real-time (SMRT) sequencing or Oxford Nanopore sequencing. They enable the sequencing of full-length transcripts and are not only capable of examining RNA abundance, but splice variants, structural variation and chimeric transcripts too.
However, the application of PacBio SMRT to single-cell sequencing has been hindered by a low sequencing capacity, which means that a single run can report on only 40–133 cells at a comparable read depth to short-read approaches. The main drawback of nanopore sequencing is its high error rate compared with both PacBio long-read and Illumina short-read sequencing (5–15% compared with less than 1%). This leads to errors in the critical barcode and UMI assignment steps and seriously challenges their applications in single-cell sequencing.
Recently, a group of researchers at the University of Oxford have developed a technology to overcome these challenges called single-cell corrected long-read sequencing (scCOLOR-seq). The novel approach provides a means for identifying and correcting errors in the barcode and UMI sequences, facilitating the standalone long-read nanopore sequencing of cells. The team evaluated the use of scCOLOR-seq and it was found that the novel approach was able to correct error-prone sequencing – it achieved over 80% recovery of reads. Although the recovery was slightly variable when using different edit distances, the application of scCOLOR-seq showed serious advantages over other sequencing technologies.
Improved long-read technology
Professor Adam Cribbs, a researcher at the University of Oxford, said: “What we’ve been able to do is to develop a practical method, allowing inaccuracies within the sequencing to be pinpointed, and then correct them. 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.”
The study demonstrated that scCOLOR-seq provides a simplified and more robust method to perform quantitative long-read transcript sequencing on large numbers of cells. This novel technology has the potential to create new opportunities within genomics and stimulate further work on understanding human disease, such as single-cell copy number variation and mutational analysis.
Professor Udo Oppermann, also a researcher at the University of Oxford, explained: “We will continue our collaborative efforts to develop innovative single-cell approaches apply this to molecular analyses. Our intention is to advance these technologies in personalised medicine approaches such as cancer diagnosis allowing rational clinical decision making.”
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