ddRAD-seq Study Explores Behavioral Roles in Speciation
A new preprint from the Hoekstra lab at Harvard makes great use of the double digest RAD-seq protocol to better understand reproductive barriers and speciation in closely related species of mice. Since it was the Hoekstra lab that gave us the ddRAD-seq method, we took notice when this preprint became available.
The paper comes from Hopi Hoekstra and Emily Delaney, a Harvard grad student who is now a postdoctoral fellow at the University of California, Davis. In “Sexual imprinting and speciation in two Peromyscus species,” the scientists describe how sexual imprinting, typically a learned trait, contributes to sexual isolation of Peromyscus leucopus, the white-footed mouse, and P. gossypinus, the cotton mouse.
One area of interest at the start of this project was determining the genetic or learned mechanisms underlying sexual isolation. The scientists “used genomic data to first assess hybridization in the wild and conclusively found that the two species remain genetically distinct in sympatry despite rare hybridization events,” they report. “We find that these mating preferences are learned in one species but may be genetic in the other: P. gossypinus sexually imprints on its parents, but innate biases or social learning affects mating preferences in P. leucopus.”
The study involved using ddRAD-seq to analyze 376 mice. In that workflow, the team used Pippin Prep to select fragments ranging from 265 bp to 335 bp. Libraries were sequenced with the Illumina platform.
“Our study supports an emerging view that sexual imprinting could be vital to the generation and maintenance of sexual reproductive barriers,” the authors conclude. “Examining the role of sexual imprinting in similar cases of speciation driven by sexual reproductive barriers will continue to expand our understanding of the role of behavior in speciation.”
For 10x Genomics Workflow, Broad Institute Uses PippinHT Size Selection
At the Broad Institute, scientist Michelle Cipicchio is part of the technology development team responsible for optimizing new methods or sample types before they’re implemented on the organization’s industrial-scale exome and whole-genome sequencing pipeline. Recently, she’s been working with the Chromium platform from 10x Genomics, and part of getting it ready for production involved implementing the PippinHT for automated DNA size selection.
The technology development team is focusing on whole genome analysis with the Chromium platform. To put the workflow through its paces, they’re running a pilot project on 450 whole blood samples for scientists conducting a large schizophrenia study.
Cipicchio began working with automated DNA size selection from Sage Science at the recommendation of 10x Genomics. “The first step in the 10x process requires the longest DNA molecules that you can acquire,” she says. Since the Broad often uses legacy samples that have gone through multiple freeze/thaw cycles, her team doesn’t have the luxury of expecting high-quality, intact DNA. “For 10x, these long molecules are really necessary and most of our samples don’t have a ton of that kind of material,” Cipicchio adds. She began using BluePippin to remove smaller fragments prior to library construction. The team evaluated four samples with and without Pippin size selection and found that they were consistently able to get longer phasing data with automated size selection. To ramp up capacity so all 450 samples can be run with size selection prior to Chromium processing, the team upgraded to the higher-throughput PippinHT platform.
Optimization work for the workflow is still underway. Cipicchio and the team have run about 100 of the 450 samples so far, so they have lots more opportunities to polish and perfect the protocol before it’s ready for production mode.
PacBio Users: Size Selection Is Essential for Generating Excellent Results
The Sage Science team was delighted to attend and co-sponsor PacBio’s annual East Coast user group meeting in Baltimore last week, particularly since there was a half-day session devoted to our favorite subject: sample prep.
There were plenty of customer presentations during the sample prep workshop, and it was great to see so many PacBio users deploying BluePippin, PippinHT, or SageELF in their sequencing workflows. Melissa Laird Smith from the Icahn School of Medicine at Mount Sinai may have put it best when she told attendees that the two most important components for PacBio sample prep are upfront quality control and size selection. The QC step, of course, evaluates sample quality and quantity to ensure that long-read sequencing is viable. Size selection allows users to really make use of their PacBio platforms by eliminating shorter fragments and letting the sequencer focus on the longest fragments available. Those are often used as seed reads to anchor assemblies, making them critical for achieving optimal contiguity. Smith said her team uses BluePippin or PippinHT to select either 10 kb – 50 kb or 20 kb – 50 kb ranges, depending on the sample.
Sonny Mark, a field application scientist manager at PacBio, also took the opportunity to introduce attendees to the SageHLS extraction and purification instrument we launched earlier this year. Designed expressly for the kind of high molecular weight DNA that single-molecule systems require, the SageHLS platform should be a nice fit for long-read sequencing pipelines. Users simply load their samples (up to four at a time) and the instrument extracts or purifies DNA fragments as long as 2 Mb. The fragments are automatically sorted by size into six collection bins. We anticipate that this product will work well for scientists studying structural rearrangements, copy number variation, haplotype phasing, and other applications for which HMW DNA is advantageous.
During the rest of the user group meeting, we thoroughly enjoyed learning about so many impressive results users have generated with their PacBio systems, from reference-grade genome assemblies to in-depth annotations. Congratulations to everyone who contributed!
Preprint Highlights Utility of Size Selection for Nanopore Studies
A recently shared preprint demonstrates the effectiveness of size-selection for nanopore sequencing, relying on the PippinHT automated DNA sizing platform for high-throughput pipelines.
“Mapping And Phasing Of Structural Variation In Patient Genomes Using Nanopore Sequencing” comes from lead author Mircea Cretu Stancu and collaborators at University Medical Center Utrecht, the University of Torino, and other institutions. In it, the scientists report results from using an Oxford Nanopore MinION to sequence the genomes of two patients with congenital abnormalities, with a focus on structural variant (SV) detection. “Long-read sequencing is breaking ground for the discovery of SVs at an unprecedented scale and depth,” they write. The team used the PippinHT system to size-select DNA libraries for the second patient prior to sequencing.
The effort, which produced the first known whole human diploid genome assemblies using the MinION, was a success. “We were able to extract all known de novo breakpoint junctions for Patient1, even at relatively low coverage,” the scientists report. For the second patient, the sequence data revealed more complexity for many breakpoint junctions. “We observed that 33.3% of the high confidence set of SVs observed in the Nanopore data could not be found in matching Illumina sequencing data, despite the use of six different variant calling methods,” they add.
The authors note that “these results highlight the feasibility to sequence clinical human samples in real-time on a low-cost device.”
Creighton Lab Boosts Yield, Sequencing Efficiency with BluePippin
At Creighton University in Omaha, Neb., Dr. Anna Selmecki’s lab explores various fungal species to understand genome instability, pathogenesis, and the acquisition of drug resistance. For these investigations, her team relies heavily on whole genome sequencing, using both the Illumina MiSeq platform and Oxford Nanopore sequencers.
However, Selmecki and her team encountered two major obstacles with their library preparation pipeline. A bead-based size-selection step was decreasing their yield and even with size selection, the MiSeq was still generating very short reads. Using AMPure magnetic beads for sizing, “we always found that we lost a huge percentage of the library,” Selmecki recalls. Even when a Bioanalyzer reported that the library fragment size was in the desired range, sequencing results were consistently shorter than expected.
While both problems stemmed from the sizing step, switching to commonly used manual gel excision was not an option. “From previous experience, I knew that cutting bands out of a gel is horrible and you still lose a lot of your library that way,” Selmecki says. She remembered from her days at the Dana-Farber Cancer Institute that colleagues had raved about an automated size selection instrument from Sage Science.
So Selmecki brought in the BluePippin sizing platform and solved both problems. Recovery is significantly better, and more precise size selection removes the small fragments that had been leading to shorter-than-anticipated MiSeq reads. “The Pippin cleaned that up a lot, ensuring that we’re only amplifying pieces that are much larger,” she says. Using BluePippin for size selection followed by bead-based purification, Selmecki and her team can easily select for insert sizes of 600 bp to 1.2 Kb for their paired-end sequencing pipeline. “We found we got better coverage across the genome,” she adds.
Selmecki’s team is already planning to expand the use of its BluePippin instrument to other molecular biology techniques, such as molecular cloning and library preparations for Oxford Nanopore sequencing. “We’re just doing everything on the Pippin,” she says.
“If people are noticing really uneven coverage across their genomes or they’re having trouble with yield during their library prep, I would recommend considering the Pippin,” Selmecki says.