IsoSeq

Iso-Seq is a long-read RNA sequencing approach designed to capture full-length transcript isoforms directly from polyadenylated RNA. Developed for use with Pacific Biosciences SMRT sequencing platforms, it enables sequencing of complete transcripts from end to end, facilitating identification of alternative splicing, transcription start sites, and polyadenylation without the need for transcript assembly from short reads.

The method relies on generating full-length cDNA molecules that retain both the 5' cap and the 3' poly(A) tail. cDNA is converted into SMRTbell libraries and sequenced on PacBio instruments. Reads are processed into circular consensus sequencing (CCS) reads with high accuracy. The Iso-Seq analysis workflow classifies CCS reads as full-length non-concatemer (FLNC) reads, clusters them to produce high-quality transcript sequences, and then collapses redundant reads to produce a non-redundant set of transcript models. Outputs are typically aligned to a reference genome or used to annotate novel isoforms in de novo transcriptomes.

Applications of Iso-Seq include refining genome annotations, discovering novel splice variants and alternative polyadenylation patterns, and producing high-confidence isoform-level models in both model and non-model organisms. Its primary advantages are the capture of complete exon–intron structures and more accurate representation of transcript diversity, reducing reliance on computational assembly from short reads. Limitations include the need for high-quality RNA input, higher cost and lower throughput compared to short-read RNA-Seq, and greater computational resources for processing. Iso-Seq remains a widely used tool for transcriptome characterization and isoform discovery.