A complete solution for high-quality genome annotation using the PacBio Iso-Seq method
The PacBio Iso-Seq method produces high-quality, full-length transcripts of up to 10 kb and longer and has been used to annotate many important plant and animal genomes. We describe here the full Iso-Seq ecosystem that enables researchers to achieve high-quality genome annotations. The Iso-Seq Express workflow is a 1-day protocol that requires only 60-300 ng of total RNA and supports multiplexing of different tissues. Sequencing on a single SMRT Cell 8M on the Sequel II System produces up to 4 million full-length reads, sufficient to exhaustively characterize a whole transcriptome on the order of 15,000-17,000 genes with 100,000 or more transcripts. Most importantly, the method is supported by a maturing suite of official and community-developed tools. The SMRT Link Iso-Seq application outputs high-quality (>99% accurate), full-length transcript sequences that can optionally be mapped to a reference genome for a single SMRT Cell worth of data in 6-9 hours. For example, the SQANTI2 tool classifies Iso-Seq transcripts against a reference annotation, filters potential library artifacts, and processes information from both long read-only and short read-based quantification. IsoPhase is a tool for identifying allele-specific isoform expression. Cogent has been used to process Iso-Seq transcripts in a genome-independent manner to assess genome assemblies. Finally, IsoAnnot is an up-and-coming tool for identifying differential isoform expression across different samples. We describe how these tools complement each other and provide guidelines to make the best use out of Iso-Seq data for understanding transcriptomes.