Fourth-generation sequencing in the cell and the clinic

Abstract

Nearly 40 years ago, DNA was sequenced for the first time. Since then, DNA sequencing has undergone continuous development, passing through three generations of sequencing technology. We are now entering the beginning of a new phase of genomic analysis in which massively parallel sequencing is performed directly in the cell. Two methods have recently been described for in situ RNA sequencing, one targeted and one untargeted, that rely on ligation chemistry. This fourth generation of sequencing technology opens up prospects for transcriptomic analysis, biomarker validation, diagnosis and patient stratification for cancer treatment.

Figure 1

Molecular steps to generate sequencing substrate in targeted and non-targeted in situ sequencing. (a) In the targeted approach, a specific or random primer is used to retrotranscribe mRNA (gray) (i), the synthesized cDNA is made single stranded and a padlock probe is hybridized to complementary target sequences on the cDNA molecule (ii, iii), leaving a short gap between the probe arms (in red). Polymerization gap filling and DNA ligation are used to circularize the padlock probe (iv), which can be then replicated via target-primed rolling circle amplification (v). (b) In non-targeted in situ sequencing, the cDNA is synthesized by random primers containing a sequencing adaptor (vi). RNA is then digested and single-stranded cDNA molecules are self-circularized (ii, iii). An external primer is then hybridized and used to prime the rolling circle amplification (RCA) reaction (ix, x). In both the approaches, the sequencing substrate consists of a rolling circle product containing a clonally amplified region of DNA (colored in red or green), which is sequenced in situ by sequencing using ligation chemistries.