AdapterLigation

Adapter ligation is a molecular biology technique in which short, synthetic double-stranded DNA sequences called adapters are covalently attached to DNA fragments. Adapters provide priming sites for amplification and sequencing and may include index sequences to enable multiplexing. They are designed to be compatible with downstream applications such as next-generation sequencing or cloning.

Adapters are ligated to DNA fragments using DNA ligases. Depending on the workflow, fragments may have blunt ends or overhangs (sticky ends). In many sequencing workflows, DNA ends are repaired to a uniform state and often tailed with a single adenine (A) to promote efficient ligation of adapters with thymine overhangs. The resulting library molecules contain adapter sequences on both ends, allowing PCR amplification and platform-specific sequencing priming.

A typical sequencing library preparation workflow involves fragmenting the input DNA, performing end repair, A-tailing, ligating adapters, cleaning up the reaction, and size-selecting for fragments within a desired length range. The library is then amplified by PCR to add full-length sequencing primer sites and, if needed, index sequences for sample identification.

Applications and considerations: Adapter ligation is central to most DNA and RNA library preparations for next-generation sequencing, including whole-genome, exome, targeted, and transcriptome sequencing, as well as small RNA libraries. Challenges include ligation bias, the formation of adapter dimers, and chimeric constructs; mitigating measures include optimizing adapter-to-insert ratios, careful size selection, and using high-fidelity enzymes. Variations exist for blunt-end versus sticky-end ligation and for RNA-based workflows.