The applications of single-cell genomics

Abstract

We all start out as a single totipotent cell that is programmed to produce a multicellular organism. How do individual cells make those complex developmental switches? How do single cells within a tissue or organ differ, how do they coordinate their actions or go astray in a disease process? These are long-standing and fundamental questions in biology that are now becoming tractable because of advances in microfluidics, DNA amplification and DNA sequencing. Methods for studying single-cell transcriptomes (or at least the polyadenylated mRNA fraction of it) are by far the furthest ahead and reveal remarkable heterogeneity between morphologically identical cells. The analysis of genomic DNA variation is not far behind. The other 'omics' of single cells pose greater technological obstacles, but they are progressing and promise to yield highly integrated large data sets in the near future.

Figure 1.

cDNA template-switching and modifications for single-cell mRNA transcriptomes. (A, left) The conventional ‘Smart-Seq’ system as applied in references (1) and (15). (B) Modifications from our group that enhance these techniques (–19). The first is the use of oligo-dT-linked beads as a priming source, which allows for reuse of the original sample several times over. The second is the use of linkers at each end of the cDNA that incorporate T3 and T7 promoters. Oligonucleotide linkers are shown in boxes. The cDNA sequence is shown in blue, and the RNA sequence is shown in red. Subscripted letters indicate a longer number of nucleotides. X equals a PCR priming sequence and Xʹ is its complement. For ‘Smart-Seq’, a common primer is used at both ends of the cDNA construct. The boxed constructs in (B) refer to the T3 and T7 promoter sequences for run-off RNA synthesis and are not to scale. V = T, G or C. N = any nucleotide. Q and S = non-promoter spacer sequences.

Figure 2.

The MALBAC system for WGA. Genomic DNA (shown as a single strand in A) is denatured and annealed to a series of random primers (8-mers) that all share a common 27 base tail (shown in red, B). Extension of these primers leads to a series of products that are all terminally tagged (in red, C). These are then put through another round of denaturation, annealing and extension (D and E). This results in extension products that have a (red) MALBAC tag at one end and its complement (shown in yellow) at the other end (F). These are annealed to form loops and remove them from the amplification reaction (G). After five rounds of this ‘quasilinear’ pre-amplification, the annealed loops are used as the substrates for conventional PCR using the MALBAC tags (the terminal red tags they all share). Adapted from reference (29).