The High Pass Plus™ gel cassette is the newest addition to the Pippin Family. As the name suggests, it is dedicated to our BluePippin “High-Pass” DNA size selection which has been a go-to method for increasing the read lengths for long-read sequencing.
High Pass size selection removes smaller DNA fragments from a sheared genomic DNA (or sequencing library) while collecting the remaining larger fragments above a tightly controlled size threshold. This way, larger molecules can be presented to the detector or droplet, and better sequencing performance can be achieved.
Long-read sequencing sample prep has been improving overall, in terms of set-up time and workflow. We decided to look at the High Pass as well to see if we could optimize the approach. To this end, we designed an entirely new gel cassette dedicated to High Pass– the High Pass Plus. We’re happy to say that we were able to cut the runtime in half and improve performance and yield.
Here’s a comparison between the standard BluePippin Cassette and the High Pass Plus:
The High Pass Plus cassette has a stocky separation column, so DNA has a shorter distance to travel. The wider column and increased taper provide higher resolution for a cleaner and more accurate size cut-off. The larger sample wells now allow a maximum load of 10ug (100% up from standard Blue Pippin cassettes). We’ve also increased the size of the elution module and surface area of the filtration membrane, bringing about improved sample recovery and reproducibility.
Here’s what we were able to accomplish, when compared to the current BluePippin standard:
• Half the run time
• Twice the loading capacity
• Better recovery and reproducibility
We offer a >15kb High Pass Plus at this time. Next up will be a >20kb and >30kb.
Blue Pippin software requirement is v6.31/6.40 CD31. Available here:
The High Pass Plus cassette is available now, order number BPLUS10, or if you’d to purchase a 3 pack (BPLUS03) to try let us know.
Last year, we posted a story about an international team of scientists who embarked on a mission with Brazilian researchers to study the dangerous mosquito-borne Chikungunya virus (“Tracking Chikungunya: New Study Traces Outbreak Path”- we were pleased to donate a Pippin Prep to the effort.)
Recently the team published a new study in Science, having deployed NGS to elucidate the epidemiology of the Yellow Fever virus in Brazil: “Genomic and epidemiological monitoring of yellow fever virus transmission potential”. According to the authors, virological surveillance requirements are to “ (i) track epidemic origins and transmission hotspots, (ii) characterize genetic diversity to aid molecular diagnostics, (iii) detect viral mutations associated with disease severity, and (iv) exclude the possibility that human cases are caused by vaccine reversion.” By sampling humans and non-human primates (NHP) across the state of Minas Gerais, the epicenter of a 2017 outbreak, the researchers analyzed how the virus spreads through space, between humans and NHPs, and the “contribution of the urban cycle”. The authors note that this type of real-time monitoring can contribute to global efforts to eliminate future epidemics – and one would assume, potentially save lives.
Our minor contribution to this work was based on outreach from Antonio Charlys Da-Costa, who had used a Pippin Prep as part of his studies at Dr. Eric Delwart’s lab at the UCSF Blood Systems Research Institute. Dr. Da-Costa, now at the Sao Paulo Institute of Tropical Medicine, was able to marshal resources from a number of other suppliers for this cause, including Illumina, Zymo Research, and Promega (surely there were others, apologies for the non-mention). Congratulations to the whole team, it’s inspiring to see such diverse institutions and agencies pulling these large-scale efforts together – and individuals like Antonio who went the extra mile.
An interesting side note, a number of other important viral genomic findings were also published during this time by a consortium of Brazilan labs in collaboration with Dr. Delwart and UCSF:
Later this week, scientists will descend on Old Billingsgate, London, for Oxford Nanopore’s annual user event. Better known as London Calling, the meeting has become famous for talks full of cutting-edge nanopore sequencing results, novel protocols, and best practices. The information exchange at London Calling has helped take nanopore sequencing technology from something only a few labs could perform well to a platform that’s now far more robust and reliable for a broad range of applications.
Sage is delighted to be attending London Calling this year. We enjoy getting to know the Oxford Nanopore users, many of whom are implementing our automated DNA size-selection tools to extract the longest possible reads from their sequencers. (For a great example, check out this new app note.) By removing the smallest fragments from libraries ahead of time, sequencers can be directed to focus on the molecules most likely to yield those record-shattering read lengths. The current record for a single continuous read is more than 1 Mb, but we’re willing to bet that this year’s London Calling users will be ready to beat it.
BluePippin users are generating longer reads from their Oxford Nanopore MinION sequencers, as demonstrated in a new app note from our colleagues at Nippon Genetics. The app note showcases a workflow used by Kazuharu Arakawa, an associate professor in the Institute for Advanced Biosciences at Keio University in Japan.
The app note describes an experiment performed on DNA extracted from the leg of a golden silk spider. Sample prep included size-selection with a BluePippin instrument to remove the shorter library fragments that would otherwise limit the amount of data produced by the MinION nanopore sequencer. As the app note points out, this step must be handled prior to library prep, during which electrophoresis-sensitive tethers are added.
According to Dr. Arakawa, “Size selection using BluePippin allowed us to remove short reads and perform effective sequence of long-chain DNA fragments, compared to using samples not size selected.”
If you’re in the Boston area, don’t miss this Friday’s one-day symposium on genomics and computational biology at the Broad Institute. Organized by the Genomic Exchange Community, this geXc (pronounced “jexy”) event will bring together speakers from academia and industry to offer updates on everything from the microbiome and data analysis to genome modeling and population studies.
Sage is proud to be a sponsor of geXc Boston. Our own Jun Zhou, a senior scientist, will be giving a talk about using the HLS-CATCH method for targeted, long-range sequencing of large genomic elements. The event is free to attend and includes giveaways of an Oxford Nanopore MinION and an Apple Watch. We hope to see you there!