Tagmentation-based single-cell genomics

Abstract

It has been just over 10 years since the initial description of transposase-based methods to prepare high-throughput sequencing libraries, or "tagmentation," in which a hyperactive transposase is used to simultaneously fragment target DNA and append universal adapter sequences. Tagmentation effectively replaced a series of processing steps in traditional workflows with one single reaction. It is the simplicity, coupled with the high efficiency of tagmentation, that has made it a favored means of sequencing library construction and fueled a diverse range of adaptations to assay a variety of molecular properties. In recent years, this has been centered in the single-cell space with a catalog of tagmentation-based assays that have been developed, covering a substantial swath of the regulatory landscape. To date, there have been a number of excellent reviews on single-cell technologies structured around the molecular properties that can be profiled. This review is instead framed around the central components and properties of tagmentation and how they have enabled the development of innovative molecular tools to probe the regulatory landscape of single cells. Furthermore, the granular specifics on cell throughput or richness of data provided by the extensive list of individual technologies are not discussed. Such metrics are rapidly changing and highly sample specific and are better left to studies that directly compare technologies for assays against one another in a rigorously controlled framework. The hope for this review is that, in laying out the diversity of molecular techniques at each stage of these assay platforms, new ideas may arise for others to pursue that will further advance the field of single-cell genomics.

Figure 1.

The anatomy of a tagmentation reaction. (A) The structure of the tagmentation adapter, which includes the double-stranded 19-bp mosaic end sequence recognized by Tn5 transposase, as well as a single-stranded overhang on the transfer strand that contains an adapter used for subsequent processing. This ssDNA overhang can be any length; however, shorter sequences improve efficiency of tagmentation. (B) The Tn5 enzyme is loaded with a mix of adapters with forward or reverse adapter overhangs. For standard tagmentation workflows, this includes a 1:1 molar ratio of the two adapter species and a 1:1 molar ratio of the total adapter content to Tn5 monomer. (C) The tagmentation reaction involves the binding of transposome complexes to the target DNA at high density, that is, one insertion every ∼500 bp. (D) Each tagmentation event results in the cleavage of the DNA backbone on both strands staggered by 9 bp. The 3′ end of the transfer strand is then covalently appended to the 5′ end of the nick in the target DNA backbone at each of the cut sites. (E) After the tagmentation, the Tn5 enzyme remains tightly bound to the target DNA and must be removed to enable end repair, where the bottom strand acts as a priming site to copy through the adapter on the transfer strand. (F) End repair results in the copying of the 9-bp region between the two cuts in the target DNA backbone, where the pair of adjacent library fragments produced by a single tagmentation event overlap by the 9-bp segment when aligned to a reference genome. In low input libraries, instances of the ends of two reads from separate, adjacent read pairs can be observed as the overlap between two separate, adjacent read pairs. (G) PCR amplification is then performed, using the adapter overhangs of the transfer strand as priming sites. The primers required for cluster generation or other means of sequencing along with optional sample indexes are appended here. (H) Sequencing is performed using primers that include the mosaic end and adapter sequence to obtain paired reads of target DNA as well as index sequences.

Figure 2.

Indexing strategies for tagmentation-based single-cell assays. (A) Physical isolation of single cells into individual rection vessels that are then lysed and processed individually using the standard tagmentation workflows. Indexed PCR on each reaction compartment enables single-cell discrimination. (B) In situ tagmentation is performed by performing the tagmentation reaction in bulk to produce a single cell's library contained within the nucleus of that cell. These preprocessed nuclei can then be subjected to indexing via several techniques, including sorting and indexing PCR in plates (arrow 1), droplet encapsulation with indexing PCR or primer extension (arrow 2), or combinatorial indexing strategies using ligation (arrow 3). (C) Tagmentation with a large set of indexed adapters corresponding to individual reaction wells enables the IST nuclei to then be pooled and redistributed for a second round of indexing, enabling single-cell discrimination using the combination of indexes. The preindexed nuclei can also be carried through additional rounds of indexing using ligation-based methods (arrow 4) or droplet-based methods to enable increased throughput (arrow 5).

Figure 3.

Transposome strategies to improve efficiency. (A) The standard tagmentation reaction randomly incorporates a mix of forward and reverse adapters. This results in 50% of the resulting molecules with both a forward and reverse adapter that can be carried through subsequent processing steps, with such fragments preferentially forming hairpin complexes rather than primer annealing during PCR and also being unable to form sequencing clusters. The remaining 50% are flanked by two forward or by two reverse adapter sequences and are not viable. This effectively caps the maximum efficiency of two-adapter tagmentation at 50%. (B) Several strategies have been developed that use single-adapter tagmentation with an alternative means of appending a reverse adapter. Three of these use tagmentation with a T7 promoter to enable linear amplification using in vitro transcription. The other two use either random priming or adapter switching strategies. Arrows indicate alternative processing workflows: (1) sciTIP-seq to obtain histone modification profiles, (2) sci-L3-WGS + RNA to capture RNA alongside DNA, (3) capture of targeted regions of the genome within the sci-L3-WGS workflow, (4) s3-WGS to capture whole-genome sequence with the s3 workflow, and (5) s3-GCC to capture both WGS and chromatin folding with the s3 workflow. (C) Tagmentation with an expanded set of adapters reduces the probability of producing fragments that terminate in the same adapter species from 50% to 1/n, where n is the number of adapter species present.