Several key features differentiate the commonly used single-cell and spatial sequencing methods. (A) In the standard single-cell RNA-seq approach, a single-cell suspension is created and passed through a microfluidic system that captures each individual cell in an oil droplet with a bead containing a unique barcode. After short-read sequencing, cells can be clustered based on gene expression into groups representing distinct cell types. Each read covers only a small section of a transcript, and there is a bias toward the ends of a transcript (most often the 3′ end). Relatively few reads cover an exon junction. (B) With long-read sequencing, a read covers all or most of a transcript, including multiple exon junctions. This enables comparisons between cell types in terms of the relative abundance of particular isoforms or RNA variables such as alternative exons. Many cells can be sequenced, but relatively few transcripts are sequenced per cell. (C) Smart-seq approaches allow for better coverage over the full length of a transcript, including over exon junctions, than with standard single-cell RNA-seq, while still relying on short reads. More molecules are sequenced per cell than in single-cell long-read sequencing. (D) When the entire cell is used as the source of RNA, most sequenced molecules are cytoplasmic and thus fully spliced. However, in single-nucleus sequencing, where only nuclear RNA is available, many molecules will be unspliced or partially spliced. Also, because some of the introns present in sequenced transcripts will have “decoy” poly(A) stretches, some reads will start there rather than at the 3′ end of the transcript. (E) In spatial methods, a tissue slice is tagged with a grid of barcodes that encode each transcript's position within the slice rather than pointing to an individual cell of origin.
