At Creighton University in Omaha, Neb., Dr. Anna Selmecki’s lab explores various fungal species to understand genome instability, pathogenesis, and the acquisition of drug resistance. For these investigations, her team relies heavily on whole genome sequencing, using both the Illumina MiSeq platform and Oxford Nanopore sequencers.
However, Selmecki and her team encountered two major obstacles with their library preparation pipeline. A bead-based size-selection step was decreasing their yield and even with size selection, the MiSeq was still generating very short reads. Using AMPure magnetic beads for sizing, “we always found that we lost a huge percentage of the library,” Selmecki recalls. Even when a Bioanalyzer reported that the library fragment size was in the desired range, sequencing results were consistently shorter than expected.
While both problems stemmed from the sizing step, switching to commonly used manual gel excision was not an option. “From previous experience, I knew that cutting bands out of a gel is horrible and you still lose a lot of your library that way,” Selmecki says. She remembered from her days at the Dana-Farber Cancer Institute that colleagues had raved about an automated size selection instrument from Sage Science.
So Selmecki brought in the BluePippin sizing platform and solved both problems. Recovery is significantly better, and more precise size selection removes the small fragments that had been leading to shorter-than-anticipated MiSeq reads. “The Pippin cleaned that up a lot, ensuring that we’re only amplifying pieces that are much larger,” she says. Using BluePippin for size selection followed by bead-based purification, Selmecki and her team can easily select for insert sizes of 600 bp to 1.2 Kb for their paired-end sequencing pipeline. “We found we got better coverage across the genome,” she adds.
Selmecki’s team is already planning to expand the use of its BluePippin instrument to other molecular biology techniques, such as molecular cloning and library preparations for Oxford Nanopore sequencing. “We’re just doing everything on the Pippin,” she says.
“If people are noticing really uneven coverage across their genomes or they’re having trouble with yield during their library prep, I would recommend considering the Pippin,” Selmecki says.