Regular readers of the Sage Science blog know that we can never resist a good methods paper. We enjoyed this publication from the phyloinformatics group at RIKEN detailing a modified protocol for generating high-quality mate-pair libraries while significantly reducing costs.
Lead author Kaori Tatsumi and colleagues focused on Illumina’s Nextera Mate Pair Sample Prep Kit, a workhorse for this application. They rely on mate-pair sequencing to improve the contiguity of de novo assemblies, particularly for non-model organisms. The Nextera kit “has significantly reduced the difficulty, preparation time and cost of preparing mate-pair libraries,” the scientists report, but “there remain opportunities to improve the efficiency and reduce costs for this preparation technique.”
The team tried several tactics to achieve this goal. They started with reducing the amount of enzyme used in the workflow, and also concocted their own homemade buffer. Tests showed that these worked at least as well as the original protocol. “The use of a reduced volume of enzyme and self-made buffer allows for a higher number of tagmentation reactions to be attempted using variable conditions,” the scientists write. “In particular, this modification, which yields larger DNA molecules, is advantageous in preparing mate-pair libraries spanning large distances (>10 kb), which tends to be hindered by low yields.”
They also swapped the manufacturer’s recommended order of the strand displacement step and the size selection step, sizing DNA first on the BluePippin and then performing strand displacement. “Reversing the order of strand displacement and size selection enables significantly smaller volumes of strand displacement polymerase and buffer for reduced amounts of size-selected DNA, which allows for a larger number of preparations (up to 3-fold) than the standard protocol,” Tatsumi et al. found. The revised process still yielded enough library volume for the rest of the pipeline.
Finally, they added more shearing steps to improve accuracy of reads recognized as mates, and adjusted the number of cycles of sequencing on an Illumina HiSeq. Compared with libraries prepared according to the original protocol, their method resulted in much longer scaffold lengths and a higher percentage of genes covered completely in the assembly. The revised protocol also costs significantly less than standard preparation.
Kudos to the RIKEN team for their hard work on a cool new method!
We always knew genomics would have a positive impact on healthcare, but as we were dreaming about tailored therapies, targeted cancer treatments, and preventive medicine based on known susceptibilities, there was one area we didn’t think about: HLA typing. It’s a slice of the medical field that recently adopted next-gen sequencing and is already seeing great results.
HLA (human leukocyte antigen) typing has been around for decades. It’s the science of using specific variants in these proteins, which regulate the immune response and vary considerably from person to person, to find the ideal match for an organ or bone marrow transplant. HLA is produced by the histocompatibility locus, a highly polymorphic stretch of the genome.
As the proteins have become better understood, scientists have added more markers to the process of determining a good match. Today clinicians will review eight to 10 markers in each donor and each patient before calculating the likelihood of a positive transplant outcome.
There is massive demand for HLA typing, which means it needs to be affordable, high-throughput, and incredibly accurate — a natural fit for NGS technologies. We’re glad to see that in recent years, well-established HLA labs around the world have begun to adopt NGS platforms for their typing pipelines with demonstrated success. As these technologies become more commonplace in the HLA community, we hope to see more accurate results that make organ transplantation safer and more successful.
We’re proud to be sponsoring a new series of podcasts from the great folks at Mendelspod, who have been producing informative interviews with interesting scientists for several years now. We don’t have anything to do with the podcasts themselves, but we think they’re an important educational tool for the biotech field.
This series focuses on challenges in sequencing, and Rod Wing, director of the Arizona Genomics Institute, is a great first participant. Wing is best known in the plant genomics community for his commitment to high-quality assemblies of important crop genomes, such as rice. His focus on these crops serves as a response to the challenge of how we’ll generate enough food to feed an expected 9 billion people by 2050 — a question he talks about in some detail in this kick-off podcast.
We have long been impressed by Wing’s mastery of large DNA fragments (many scientists rely on his BAC resources), and in this podcast he speaks about the importance of long-read sequencing in establishing gold-standard genome assemblies. De novo sequencing is critical when it comes to cataloguing the full genetic variety of organisms, such as various rice strains, Wing tells Mendelspod’s Theral Timpson.
The first half of the podcast focuses on the supercrops that Wing and others are trying to breed. Then around the 19 minute mark, Wing begins talking about the limitations of assemblies based on short-read sequence (they’re full of hot air, he says) and the need for higher-quality assemblies.
We’re already looking forward to the next episode, and hope that you have the time to listen as well.
We can’t wait for ASMS, the annual meeting of the American Society for Mass Spectrometry. This year, the mega event will be held in St. Louis from May 31st to June 4th and our team will be on the scene again.
What we enjoy most about ASMS is the creativity. Proteomic experts aren’t ones to be limited by technology, so ASMS more »
We’re already looking forward to the 115th General Meeting of the American Society for Microbiology. It’ll be held in New Orleans this year from May 30th to June 2nd, giving us a great excuse to stop by Café du Monde and fill up on their world-famous beignets. Then we’ll be heading to the city’s enormous convention center for ASM, along with 8,000 other attendees.
We go to lots of scientific conferences each year, but ASM is the only one that so effectively freaks us out. (We’re just now recovering from the chikungunya virus presentations we saw at this meeting last year.) ASM is where you go to learn what’s living on the armrest of the airplane seat, in the depths of the jungle, and on your keyboard. It’s not for the faint of heart.
But it is informative and thought-provoking. We’re eager for the opening session, in which Pieter Dorrestein from the University of Californa, San Diego, will give a talk with the intriguing title “The Social Molecular Network of Microbes.” Samantha Joye from the University of Georgia will present data on the microbial response to the Deepwater oil spill, and New York University’s Martin Blaser will talk about “Our Missing Microbes.”
In the last several years, it’s been a thrill to see how much next-gen sequencing technologies have shaped what’s possible in microbiology. The shift to high-throughput, rapid platforms that can produce finished microbial sequences with minimal effort has opened all sorts of doors in this field. As NGS becomes a workhorse of this community, automated DNA size selection has become a critical addition to these sequencing pipelines as well.
Sage Science will be in booth #766, so please stop by to say hello! We’d be happy to talk to you about how more accurate DNA sizing can improve your NGS-based microbial experiments.