strandspecific

Strand-specific sequencing, also called strand-specific or directional sequencing, refers to experimental approaches in which sequencing data retain information about the originating strand of the genome or transcript. In RNA sequencing, this means that each read can be assigned to either the sense or antisense strand of the gene from which it was transcribed.

In many conventional RNA-Seq protocols, reads are not linked to a particular strand, which can obscure antisense transcription and complicate the interpretation of overlapping genes. Strand-specific methods improve accuracy in gene annotation, expression quantification, and the study of antisense transcripts, alternative splicing, and overlapping transcription units.

Common strategies for achieving strand specificity include (1) the dUTP second-strand marking method, where the second cDNA strand is marked and excluded from amplification to preserve the orientation of the first strand, and (2) directional or ligation-based library preparation methods that attach adapters in a way that preserves strand information. Various commercial kits implement these principles, often using polyA selection or ribosomal RNA depletion as a precursor to library construction.

Applications of strand-specific sequencing include improved annotation of complex transcriptomes, more accurate measurement of antisense and sense transcript levels, and better resolution of isoform-specific expression. While strand-specific protocols can reduce ambiguity and bias, they may require more input material, incur higher costs, and introduce methodological biases that researchers must account for in analysis.

See also: RNA sequencing, directional library preparation, antisense transcription, transcriptomics.