adapterligering

Adapterligering, or adapter ligation, is a molecular biology technique used to attach short double-stranded DNA adapters to DNA fragments. The adapters provide known sequences for primer binding and enable the fragments to be amplified and sequenced on next-generation sequencing (NGS) platforms. This step is central to preparing sequencing libraries from genomic DNA, cDNA, or other nucleic acids.

In a typical workflow, DNA is first fragmented to a suitable size. The ends of these fragments are then repaired and, depending on the protocol, an A overhang or blunt ends are created. DNA adapters, which contain platform-specific sequences and sometimes sample indexes (barcodes), are ligated to the fragment ends using a DNA ligase. After ligation, size selection removes unwanted fragments and adapter dimers. A PCR amplification step enriches for fragments that carry correctly ligated adapters and adds additional index sequences if needed. The resulting library is ready for sequencing.

Adapters are designed to be platform-specific. For example, Illumina-compatible adapters include sequences that bind to the flow cell and primer sites for sequencing; in some protocols, dual indexing is used to reduce misassignment of reads. There are variations in the ligation strategy, such as blunt-end ligation or ligation after an A-tailing step to create complementary overhangs.

Applications of adapter ligation span whole-genome sequencing, targeted sequencing (including exomes), RNA-seq (via cDNA libraries), ChIP-seq, and other library preparation workflows. Challenges include the potential formation of adapter dimers, biased representation of fragments due to ligation efficiency, and the introduction of artifacts or chimeric sequences if ligation occurs between fragments.