Highly accurate barcode and UMI error correction using dual nucleotide dimer blocks allows direct single-cell nanopore transcriptome sequencing

Abstract

Droplet-based single-cell sequencing techniques have provided unprecedented insight into cellular heterogeneities within tissues. However, these approaches only allow for the measurement of the distal parts of a transcript following short-read sequencing. Therefore, splicing and sequence diversity information is lost for the majority of the transcript. The application of long-read Nanopore sequencing to droplet-based methods is challenging because of the low base-calling accuracy currently associated with Nanopore sequencing. Although several approaches that use additional short-read sequencing to error-correct the barcode and UMI sequences have been developed, these techniques are limited by the requirement to sequence a library using both short- and long-read sequencing. Here we introduce a novel approach termed single-cell Barcode UMI Correction sequencing (scBUC-seq) to efficiently error-correct barcode and UMI oligonucleotide sequences synthesized by using blocks of dimeric nucleotides. The method can be applied to correct either short-read or long-read sequencing, thereby allowing users to recover more reads per cell and permits direct single-cell Nanopore sequencing for the first time. We illustrate our method by using species-mixing experiments to evaluate barcode assignment accuracy and evaluate differential isoform usage and fusion transcripts using myeloma and sarcoma cell line models.

Highly Multiplexed Single-Cell Full-Length cDNA Sequencing of human immune cells with 10X Genomics and R2C2

“…Instead of sequencing transcript fragments, long-read sequencing methods in the form of Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) are now capable of sequencing comprehensive full-length transcriptomes [16][17][18][19]. These methods have now been used to analyze single cell cDNA pools generated by different methods, both well- [20][21][22] and droplet-based [23][24][25], enriching the information we can extract from single cells experiments. However, for the analysis of high-throughput droplet-based experiments with long reads, short-read data are still required for interpreting experimental data [26] or enabling the identification of cellular and molecular identifiers in low-accuracy ONT reads [24].…”

Discovering Cellular Mitochondrial Heteroplasmy Heterogeneity with Single Cell RNA and ATAC Sequencing

“…Additionally, longread sequencers typically obtain fewer reads per UMI than short-read sequencers further compounding the issue of accurate UMI usage. Efforts are being made to address these obstacles [41][42][43]. Smart-seq3 has recently been developed as a full-length UMI protocol, though it has not yet attained widespread usage [44].…”