More and more scientists are using their PacBio systems for transcriptome studies, generating full-length isoforms with the Iso-Seq method. The number of novel transcripts discovered and the implications for alternative splicing are a not-so-subtle reminder that we still have a lot to learn about gene expression.
Full RNA transcripts have lengths up to 10kb, with the largest proportion typically falling in the 3-5 kb range. Since SMRT sequencing can read the transcript from beginning to end, PacBio recommends binning the transcripts into four size ranges for comprehensive isoform surveys; 0.8-2 kb, 2-3 kb, 3-5 kb, and 5-10 kb.
PacBio provides two template preparation protocols that feature our DNA size selection instruments: a BluePippin guide and a SageELF guide. The BluePippin collects a single size fraction from each of four samples per run, while the SageELF collects 12 contiguous size fractions from a sample and can process one or two samples per run.
Here are a few details to illustrate the differences between the platforms.
- Also validated for use with long-read and Roche/NimbleGen SeqCap template protocols
- Simplified workflow to collect one fraction from each sample
- Size cut-offs are more accurate and reproducible
- Requires >5 ug of starting DNA
- Final library size bins have more continuous overlapping, improving bioinformatics analysis
- More user flexibility for combining the size bins
- Unused fractions can be recovered and saved
- Requires 3-5 ug of starting DNA
We’re pleased that these two platforms have been helpful in the Iso-Seq workflow. A newly released 10-40 kb fractionation protocol for the SageELF should make it even more useful for long-range pipelines.
If you’re a PacBio user interested in trying out the SageELF for Iso-Seq size selection, let us know.