Tutorial on the Iso-Seq Method: Applications, Protocol, and Experimental Design
Wednesday, May 6, 2015
If you missed our recent webinar on isoform sequencing with the PacBio® platform, we’ve made the full recording available for on-demand access. “Iso-Seq™ Method: Sample Prep and Experimental Design for Full-Length cDNA Sequencing” offers an overview of the application, along with specific sample prep tips, factors to consider when designing an experiment, and suggestions about what kinds of projects can take advantage of this method.
Hosted by our own Tyson Clark, the webinar begins with a look at why it’s important to capture full-length transcripts. There are known human genes that have very different functions depending on which splice variant is expressed. With alternative splicing so critical to genome function — Clark noted one Drosophila gene that can make more than 38,000 isoforms — scientists who miss the full transcript aren’t seeing the full picture of gene activity. Single Molecule, Real-Time (SMRT®) Sequencing is the only technology that enables complete views of these isoforms, from poly-A tails to 5’ ends, without assembly.
The Iso-Seq method can be used for a number of projects. Transcript identification and annotation allows users to detect alternatively spliced isoforms. Targeted sequencing can be used to show, for example, which isoforms are enriched in which tissues. Another application is normalization; the approach can be used to reduce the representation of highly expressed genes to increase the diversity of transcripts observed per SMRT Cell.
Clark walks viewers through the steps of an Iso-Seq experiment, from converting RNA to cDNA, amplification, size selection, cleanup, and sequencing. While it is possible to perform Iso-Seq analysis with as little as 5 ng of RNA input, 50 ng is the recommended minimum, and input can go as high as 1 µg. Clark recommends using RNA with a RIN score of at least 6.
He highlighted two size-selection instruments from Sage Science — the BluePippin™ and SageELF™ systems — that fit well in the PacBio workflow for isoform sequencing and help users generate reads from even the longest transcripts in their samples. Size selection allows for more even representation across cDNA of different size ranges, since smaller fragments may load preferentially on the sequencer. Clark suggests running a Bioanalyzer trace of your sample up front to determine the range of sizes if you don’t have a good sense of this already; this can inform whether to perform size selection or which sizing approach to use. (With sizing, the sample prep and sequencing process generally takes about three days.)
When designing your experiment, Clark recommends considering how many SMRT Cells you’ll need; assume that each will generate 20,000 to 25,0000 full-length transcript sequences (slightly fewer for especially large cDNA fragments). For gene-specific isoform characterization or very targeted interrogations, a single SMRT Cell may suffice. For a comprehensive survey of full-length isoforms across a transcriptome with several size fractions, you might need 12 to 16 SMRT Cells.