A new publication in Current Protocols in Human Genetics offers a thorough description of library preparation steps for sequencing with Illumina instruments. “Improved Protocols for Illumina Sequencing” comes from senior author Harold Swerdlow at the Wellcome Trust Sanger Institute and colleagues.
The detailed steps provided in the publication will “make the sequencing process more reliable in a high-throughput environment, reduce amplification bias, narrow the distribution of insert sizes, and reliably obtain high yields of data,” the authors note. The protocols described come from years of optimization studies at Sanger and represent the standard sequencing pipeline now used at the institute.
As many of our customers with Illumina sequencers know, size selection is an important part of the sample prep process. “Cluster amplification is a relatively inefficient process,” the scientists write. “Larger fragments [above 500 bp] up to 1,000 bp will cluster, but with increasingly lower efficiency and lower yield.”
Swerdlow and his colleagues use a variety of size-selection tools; when they need very tight sizing or are concerned about the generation of chimeric templates from the PCR step, they rely on our technology to perform accurate sizing. “Where a very precise/narrow size distribution is necessary, we recommend using Sage Science’s Pippin Prep,” they write. This figure from the publication shows why:
The scientists note that even in cases where especially narrow sizing is not necessary, performing a second size selection step with Pippin after running PCR is important for reducing chimeras. “This additional size selection reduces the incidence of chimeras from ∼5% to 0.02%,” they report.
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.
Between Ion World and ASHG, last week was a conference frenzy here in Boston. The Sage Science team had a great experience at both events — it was wonderful getting to talk to so many scientists in the sequencing field, including many of our customers.
At Ion World, we shared our size selection approach with researchers interested in amplicon sequencing; this has been a natural fit for users of PGM and Proton. The two-day meeting was filled with excellent talks, including technology updates from Life Technologies. The advent of a new amplification step will help reduce sample prep time and cut the overall turnaround from more than 12 hours to less than 8, according to customer Dagan Wells from the University of Oxford. The Ion workflow continues to recommend the use of Pippin sizing for most efficient loading and precise assembly results.
No sooner had we broken down our setup at Ion World than we were assembling our full booth for ASHG’s enormous exhibit hall. This was the first conference where we got to display the newest member of the Sage Science product portfolio, the upcoming Sage ELF (that’s short for Electrophoretic Lateral Fractionator – expect it in January).
This tool can take 12 contiguous fractions of a single sample, making it ideal for clinical and research pipelines running precious samples. It will allow users to prepare multiple libraries from each sample — for example, a long-insert library for PacBio, plus shorter-insert libraries for Ion Torrent or Illumina sequencing — and truly maximize the information that can be gleaned from a single sample.
Much of the excitement at ASHG related to resolving complex genetic structures, with lots of attention paid to the new human reference assembly coming soon from the Genome Reference Consortium. Plenty of large-scale studies were presented during ASHG, often featuring thousands of exomes or hundreds of genomes — confirmation that the field is ramping up to high-powered studies more likely to help determine the genetic root of rare and even common diseases. We are proud to see our technologies are helping in many of these important projects.
Thanks to everyone who stopped by our booths at Ion World and ASHG!
The annual meeting of the American Society of Human Genetics will be taking place a hop, skip, and a jump from our office — and we can’t wait! It’s one of the biggest genetics meetings on the calendar, and this year some 7,000 people will assemble at the Boston Convention and Exhibition Center to hear about cutting-edge research, new technologies, clinical studies, and more.
Sage Science will be participating at ASHG and we hope to connect with you. For information on two technologies we’ve got in development, check out our poster in the Bioinformatics & Genomic Technologies section (#1646F). In it, we introduce the high-throughput Pippin — a response to many customers who have asked for the ability to run more samples — which can process up to 24 samples per run. We also preview our new Sage ELF, which fractionates a single genomic sample into 12 contiguous DNA fractions. Both systems are designed to take advantage of better library chemistries that have reduced the DNA input required for a sequencing run and are well suited to researchers working with precious samples.
We’ll also be in the exhibit hall at booth #1104 near the front entrance. Stop by to tell us about your research and find out how automated DNA size selection from Sage can help. Looking forward to seeing you there!
A paper that came out in the Journal of Innate Immunity this summer reports that a person’s genotype affects which microbes will colonize his or her skin, which in turn may alter that person’s defense mechanisms against pathogenic organisms.
“Skin Microbiome Imbalance in Patients with STAT1/STAT3 Defects Impairs Innate Host Defense Responses” comes from a team of scientists in Boston and The Netherlands. Lead author Sanne Smeekens, from Radboud University Nijmegen Medical Center, and her collaborators investigated patients with immunodeficiencies linked to mutations in STAT1 and STAT3 that increase susceptibility to skin and mucosal infections, particularly from fungal pathogens or Staphylococcus aureus.
To determine the implications of these mutations in microbiome and host defense, the scientists compared skin and oral samples from several patients with age-matched healthy controls. Microbial colonies were assessed with 16S rRNA sequencing, performed on Illumina MiSeq after size selection with Pippin Prep.
The team found that immunodeficient patients’ microbiomes contained more Gram-negative bacteria (particularly Acinetobacter) and less Corynebacterium than their healthy counterparts. Functional studies revealed that the difference in microbiome composition leads to an inhibited immune response to Candida albicans and S. aureus in these patients.
“These data in patients with immunodeficiencies prove that the microbiome can influence host defense and could open the possibility of microbiome-based adjuvant therapy in patients with immune defects,” the authors conclude.