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Fig. 1 | Genome Biology

Fig. 1

From: Single-cell RNAseq for the study of isoforms—how is that possible?

Fig. 1

Single-cell mRNA sequencing methods and sources of mRNA variation. a Methodological approaches to single-cell isoform studies. The combination of library preparation and sequencing technologies yields three distinct methods to capture isoform diversity. UMI-based methods are limited to sequencing of the 3′ (or 5′ end), which enables usage of UMIs to capture efficiently PCR bias in addition to early cell barcoding, even if they are particularly suited to quantify expression at the gene level. Smart-based methods produce short reads across the entire transcript length, although they require late cell barcoding (barcodes inserted in tagmentation), cannot accommodate UMIs, and the reads might be difficult to assign unambiguously to an isoform. Single-molecule sequencing allows sequencing of each transcript molecule in a single read and provides full isoform connectivity, although it suffers from a high prevalence of sequencing errors. b Sources of transcript variation that yield alternative isoforms and their position along the transcript. When compared with a reference isoform (for convenience, that including all exons, no introns and the complete UTRs), alternative TSSs (transcription start sites) and TTSs (transcription termination sites) are generated during the transcription process by shortening of the UTRs. Processing of the pre-mRNA eliminates or retains introns and exons, adding variability to the isoforms that can be generated from the gene. In addition, more than one event can simultaneously be present in the same isoform, and consequently isoform diversity will increase with the number of possible combinations of AS events. Alt. alternative, RT reverse transcription, UMI unique molecular identifier

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