New Resources: App Notes for Mate-Pair and Long-Read Sequencing with BluePippin

We’ve got some new application notes to share that will be particularly handy for BluePippin customers running mate-pair libraries or sequencing with the Pacific Biosciences platform. Many thanks to our distribution partner, Nippon Genetics, for making this great information available to the community.

In one app note, data provided by Dr. Yoshitoshi Ogura and Dr. Yasuhiro Gotoh from the University of Miyazaki in Japan demonstrate the use of BluePippin in a mate-pair library workflow with Nextera tagmentation. They prepared libraries for six strains of bacteria and used BluePippin to extract 8 Kb fragments. The scientists had previously used manual gel extraction, but found it to be time-consuming and troublesome. They report that BluePippin significantly reduces the amount of time required while delivering high-quality sizing results. Illumina’s mate-pair guidelines already suggest using Pippin Prep for size selection, and we’re glad to see this work validating the use of BluePippin as well.

The other two app notes cover studies conducted to assess the value of BluePippin size selection for achieving longer subreads with the PacBio RS II sequencer. BluePippin has been quite popular with PacBio customers because it can remove short fragments from libraries, focusing sequencing efforts on the longest fragments. This process not only increases average read length, but also boosts instrument throughput.

In one project, Dr. Yasuhito Arai at Japan’s National Cancer Center Research Institute used BluePippin’s high-pass mode to remove fragments smaller than 7 Kb from a library of human genomic DNA. Results were assessed with the Pippin Pulse, our pulsed-field gel electrophoresis product that quickly checks the size of long DNA fragments. According to the study, BluePippin selection offered a real improvement: libraries built without sizing resulted in an average subread length of 2,675 bp; with BluePippin, that average increased to 4,714 bp, an improvement of 76 percent.

For the other project, a scientist from the Okinawa Institute of Advanced Sciences in Japan built three libraries of bacterial DNA: one with no size selection; one selected for fragments 4 Kb and larger; and one selected for fragments 7 Kb and larger. Sequencing was performed using PacBio’s P5-C3 chemistry. Results were checked on both the Pippin Pulse and a Fragment Analyzer from Advanced Analytical. Both evaluations demonstrated that the library made without size selection included a number of short fragments, while the 4 Kb library reduced and the 7Kb library removed short fragments. Compared to the library with no size selection, the 7 Kb library yielded a 3.3-fold increase in average subread lengths (from 2,060 bp to 6,671 bp); the amount of data per cell also increased by 1.9-fold. According to the scientist, BluePippin is effective and essential for obtaining long reads.

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