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.
