PacBio User Meeting: Size Selection for Best Results

Last month we got to attend PacBio’s user group meeting in Palo Alto, Calif. Sage Science co-sponsored the event, as we have in the past, because the PacBio community is doing some extraordinary work with size selection and we’re always eager to learn more about what they’ve accomplished.

This year, one of the speakers gave a presentation on how optimizing size selection and library prep can improve PacBio sequencing results. David Moraga Amador, scientific director of the NextGen DNA Sequencing core at the University of Florida in Gainesville, gave a terrific view of life in the core lab, where top-notch scientists are always trying to get that extra performance out of a less-than-perfect sample. (He got sympathetic laughs from the audience when he showed examples of some of the most challenging samples his users have handed off to him — we could tell there were plenty of core lab folks at the event!)

Moraga Amador’s talk, “The impact of optimum library size-selection on PacBio sequencing results,” covered a few sample prep methods, including the use of our BluePippin to maximize read length and improve sequencing yield. He emphasized the up-front protocols because that’s how his core facility manages to turn suboptimal samples into libraries that will generate solid sequencing results with PacBio.

He offered one example that was really interesting to us, based on a plant DNA sample where his team prepared half the library with BluePippin and the other half with AMPure beads to generate a 20 Kb library for PacBio sequencing. An analysis of the libraries prior to sequencing reported that they were virtually indistinguishable, Moraga Amador said. But after sequencing results from each library were compared, the BluePippin-selected library had yielded significantly longer reads. The final tally: nearly 60 percent of reads from the BluePippin-selected library were longer than 4,500 bases, while only about 25 percent of reads from the AMPure library were in that category.

Moraga Amador also noted that BluePippin is handy for the Iso-Seq method with PacBio because it’s important to feed sequences of fairly uniform length into the instrument. He size-selects into three or four groups, making sure that each group has fragments of similar size, to boost the efficiency and effectiveness of isoform sequencing.

We were glad to hear the instrument is performing so well at the Florida core lab, and it was a real treat to attend the user group meeting. We look forward to learning more at the next PacBio user event!

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