Abstract
Here we describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. Barcodes and unique molecular identifiers are synthesized using dimeric nucleotide building blocks that allow error detection. We illustrate the use of the method for evaluating barcode assignment accuracy, differential isoform usage in myeloma cell lines, and fusion transcript detection in a sarcoma cell line.
Highlights
We describe single-cell corrected long-read sequencing, which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells
To overcome the low base-calling accuracy of Oxford Nanopore sequencing, here we describe single-cell corrected long-read sequencing, in which the barcode and unique molecular identifier (UMI) regions of the oligonucleotide-barcoded RNA-capture microbeads are synthesized using homodimeric nucleoside phosphoramidite building blocks (Supplementary Fig. 1), which provides a means for sequencing-error detection and correction of the barcode and UMI (Fig. 1a)
Barcodes without errors were identified on the basis of nucleotide pair complementarity across the full length of the barcode. These accurate barcodes were used as a guide to correct the remaining erroneous barcodes. We show that this strategy is capable of correcting erroneous barcodes with a high sequencing error rate, with 96% of barcodes recovered with a barcode sequencing error rate of up to 10% (Fig. 1c)
Summary
We describe single-cell corrected long-read sequencing (scCOLOR-seq), which enables error correction of barcode and unique molecular identifier oligonucleotide sequences and permits standalone cDNA nanopore sequencing of single cells. To overcome the low base-calling accuracy of Oxford Nanopore sequencing, here we describe single-cell corrected long-read sequencing (scCOLOR-seq), in which the barcode and unique molecular identifier (UMI) regions of the oligonucleotide-barcoded RNA-capture microbeads are synthesized using homodimeric nucleoside phosphoramidite building blocks (Supplementary Fig. 1), which provides a means for sequencing-error detection and correction of the barcode and UMI (Fig. 1a). The dimer-correction approach was evaluated by measuring the proportion of human, mouse and mixed species cells identified following increased edit distances (that is, the Levenshtein distance) between the error-sequenced and the accurately sequenced barcodes (Supplementary Fig. 5).
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