Several papers have come out recently showcasing the use of our Pippin Prep with the PGM from Ion Torrent. We’re offering a synopsis of three of them below as a good way to demonstrate how different users are taking advantage of pairing these technologies.
“Semiconductor-based DNA sequencing of histone modification states,” a publication in Nature Communications, reports the use of Pippin and PGM for ChIP-seq profiling of tumor tissue. Lead author Christine Cheng from the Broad Institute and her collaborators at several institutions optimized sample preparation protocols for the generation of ChIP-seq libraries on the PGM. Two key challenges existed for implementing ChIP-seq with Ion Torrent, the authors note: low DNA input levels and the PGM’s need for a tight size range of DNA fragments. “To address the wide size range of ChIP DNA, we first tested a standard enzymatic DNA-shearing method that is routinely used with Ion Torrent genomic libraries, but failed to generate usable ChIP-Seq libraries,” the scientists write. After switching to automated size selection using Pippin Prep, they add, “we successfully created libraries for 32 of 36 samples attempted” — a nearly 90 percent pass rate. They also managed to use sub-nanogram levels of DNA to generate libraries, getting down to 0.4 ng as the starting point for building successful libraries. Cheng et al. demonstrate that results from the Ion Torrent ChIP-seq protocol were comparable to results from an Illumina workflow.
A paper from PLoS One entitled “Mitochondrial Sequence Variation in African-American Primary Open-Angle Glaucoma Patients” comes from University of Pennsylvania scientists led by David Collins. Variation in mitochondrial DNA has previously been implicated in this form of glaucoma; in this project, the authors used deep sequencing of mtDNA from patients and controls to determine whether changes in mtDNA explained the disease. Using a PGM, they sequenced 22 African-American patients with this type of glaucoma and 22 age-matched controls, finding that the original theory could not be confirmed. The number of novel variants found in the patients’ mtDNA did not differ significantly from those found in the controls, and almost all variants had been previously reported. Libraries were generated with Ion Xpress kits and adapters and then size selected with Pippin Prep. The scientists conclude, “Although it is possible that mitochondrial genetics play a role in African-Americans’ high susceptibility to POAG, it is unlikely that any mitochondrial respiratory dysfunction is due to an abnormally high incidence of novel mutations that can be detected in mtDNA from peripheral blood.”
In a paper published in ACS Chemical Biology (“Genetic Basis for the Biosynthesis of the Pharmaceutically Important Class of Epoxyketone Proteasome Inhibitors”), scientists from California and Germany teamed up to study genes that encode for the biosynthesis of an important component of certain proteasome inhibitors used for cancer treatment. Lead author Michelle Schorn and her colleagues used the PGM to sequence gene clusters in two microbial strains, reporting the first successful characterization of a region producing natural peptidyl-epoxyketones. For this project, libraries were prepared from genomic DNA sheared to 100 bp to 250 bp, after which sequencing material was separated and extracted using Pippin Prep. “With this study, we have definitively linked epoxyketone proteasome inhibitors and their biosynthesis genes for the first time in any organism, which will now allow for their detailed biochemical investigation,” write Schorn et al.