sequencingbyligation

Sequencing by ligation (SBL) is a DNA sequencing method that relies on the enzyme DNA ligase to join short labeled oligonucleotide probes to a primer that has annealed to a template. The sequence is determined by detecting which probes ligate to the primer in successive cycles, with the identity of the interrogated base revealed by the probe’s label. After each detection step, probes are removed or inactivated to allow the next cycle to interrogate a different position.

In typical SBL workflows, a library of probes complementary to the target region is organized so that each probe reports on a specific base at a defined position relative to the primer. Probes are fluorescently tagged and designed so that ligation occurs only when there is an exact match at the ligation junction. The detection of fluorescence indicates the identity of the base, and cycles are repeated with different probe sets to read successive bases. Some implementations use shifts in the reading frame or additional cycling strategies to cover longer reads.

A prominent example of sequencing by ligation is the SOLiD system, which employs a dual-base (two-base) encoding scheme in color space. In color-space sequencing, each color corresponds to a pair of bases, providing internal cross-checks and error-detection capabilities during base-calling. The resulting reads are decoded into the standard base space during data processing.

Advantages of SBL include high accuracy due to ligation specificity and built-in error-checking in certain encodings. Limitations involve shorter read lengths, more complex chemistry, and data analysis challenges compared with sequencing-by-synthesis platforms. As sequencing technology evolved, SBL contributed to early next-generation sequencing developments and informed subsequent innovations, even as other approaches became more prevalent for many applications.