New hyRAD Method Expands Use of RAD-seq to Degraded Samples
Since RAD-seq was first developed, we’ve seen a number of new versions and approaches from an enthusiastic scientific community. The latest was recently published in PLoS One and demonstrates a RAD-based method suitable for analyzing degraded DNA, an essential step for studying samples stored in museum and other collections.
“Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens” comes from lead authors Tomasz Suchan and Camille Pitteloud at the University of Lausanne and their collaborators in Russia, Poland, and the UK. The project was launched to overcome the challenges of using traditional RAD-seq methods, which require longer DNA fragments than are typically available in museum samples. “Museum collections … have not necessarily ensured optimal conditions for DNA preservation,” the authors write. “As a result, many museum specimens yield highly fragmented DNA — even for relatively recently collected samples, limiting their use for molecular ecology, conservation genetics, phylogeographic and phylogenetic studies.”
Their solution is a method called hyRAD, for hybridization RAD, which starts by using double-digest RAD-seq to produce DNA fragments from fresh samples of the species of interest. Those fragments then become capture probes for use with the degraded DNA samples. “Our method thus combines the simplicity and relatively low cost of developing RAD-sequencing libraries with the power and accuracy of hybridization-capture methods,” the team reports. “This enables the effective use of low quality DNA and limits the problems caused by sequence polymorphisms at the restriction site.”
The scientists tested this protocol on eight samples of Lycaena helle butterflies, followed by a validation project on 49 samples of the Palearctic grasshopper Oedaleus decorus. Like other RAD methods, they used Pippin Prep for size selection prior to sequencing.
“Not relying on the presence of restriction site, the method presented here should be also useful for broader phylogenetic scales, allowing sequencing homologous loci from more divergent taxa, which would not be possible to retrieve using classical RAD-seq approaches,” the scientists conclude.
AGBT in Review: Long-Range Data for Better Genome Insight
AGBT is behind us, which means the Sage Science team is officially back to the land of fleece and flannel. We had a great time at the conference and especially enjoyed seeing all the attendees making the most of our selfie sticks on the dance floor at the closing party!
The final AGBT sessions were every bit as interesting as the rest of the meeting. Nick Loman’s talk describing the use of Oxford Nanopore MinIONs during the recent Ebola epidemic in West Africa was an amazing glimpse of the kind of field-based sequencing we’ve dreamed of for a long time. His observation that the weak link in the system was the need for a constant Internet connection (required for the sequencer’s base-calling software) underscores the basic logistical challenges we face in achieving our ultimate goal of being able to sequence anything, anywhere, anytime.
The presentation from HudsonAlpha’s Shawn Levy continued the trend of 10x Genomics data, one of the major themes of this year’s AGBT. His emphasis on the importance of phasing and of finding complex events and structural variants mirrors a growing recognition in the community that short-read data will have to be supplemented by other data sources — be it long-read sequencing, Hi-C data, synthetic long reads, genome maps, or something else — for maximum benefit. Dovetail Genomics, which uses a Hi-C approach, was mentioned in several talks at the conference and really seems to be gathering steam in the field.
Normally we’d have a whole year to rest up for the next AGBT, but this September the organization will host its inaugural precision health meeting. We’re eager to see the speaker list and agenda, and maybe even get to experience the Scottsdale, Ariz., meeting in person!
AGBT: The All-Nighter Parties Haven’t Broken Us Yet
We may be a little bleary-eyed, but so far we’re surviving the Super Bowl of genomics, better known as AGBT. Last night we had a blast co-sponsoring a party out on the golf course with PacBio, and we’re glad so many people came out to eat, drink, and mingle.
The quality of scientific talks alone is enough to distinguish AGBT from other genomics conferences; this year’s slate of presentations has been no exception. We enjoyed Matt Sullivan’s talk in the opening plenary session and were delighted to see that he’s still using that carefully honed NGS pipeline for low-input samples in his new lab at Ohio State.
In a talk from the Joint Genome Institute’s Ji Lee, attendees got a nice glimpse of Oxford Nanopore’s sequencing technology in a mini-metagenomics experiment. Lee said her team regularly gets about 500 Mb of 2D sequence per flow cell and that low-input samples have provided sufficient yield for sequencing. They modified the library prep method, adding size selection with BluePippin or PippinHT after shearing to generate 20 Kb libraries — a significant boost from the median read lengths they were achieving before.
We also enjoyed the PacBio workshop yesterday, where scientists shared a number of great projects for which SMRT Sequencing had made a considerable difference in assembly quality. The event focused on human biomedical sequencing applications, so we heard some really nice examples of how long-read sequencing has provided insights for infectious disease, cancer, drug metabolism profiles, and even induced pluripotent stem cells. PacBio users have been deploying many of our automated DNA size selection tools to achieve higher average read lengths for years now, and it’s great to see these ultra-long reads making such a difference.
And now it’s back to the conference. One more day of sessions, and then we can go home and sleep for three straight days!
All Aboard for AGBT!
We can’t wait for the annual Advances in Genome Biology and Technology conference next week, but we have to keep reminding ourselves not to go on autopilot this year. For the first time since the conference began in 2000, AGBT won’t be taking place in Marco Island — this year we’re heading to sunny Orlando and wondering how many attendees will sneak out for a spin on the local Quidditch pitch.
This year’s event promises the usual great talks from highly respected scientists, with names like Sean Eddy, Harold Varmus, David Haussler, and Debbie Nickerson dotting the agenda. We’re particularly looking forward to a talk from Matthew Sullivan in the opening session; he conducted a pivotal study on DNA size selection a couple of years ago that helped demonstrate the importance of sizing in the NGS workflow. Sullivan studies viral populations in the ocean and will no doubt have a really interesting story to share with AGBT attendees. We’re also eager to hear from Katia Sol-Church on day two of the conference. Sol-Church, a leader in the core lab community, will be speaking about the clinical work her lab has gotten involved in.
The Sage team will be glued to the sessions but eager to meet old friends and old-friends-to-be at the many networking events. We’ll also be co-sponsoring a party with PacBio on Friday evening at the golf course (the Fairway Lawn, in Marriott lingo). Join us for some drinks, swag, and a great time!
ChIA-PET Study Demonstrates Effect of Genome Folding on Gene Expression
For a recent publication in Cell, scientists used the SageELF whole-sample fractionation platform to perform size selection prior to cDNA sequencing on the PacBio system. The Iso-Seq method allows PacBio users to generate full-length transcripts, and SageELF makes it easier to pool size fractions and include the exact size range of interest for the study.
In this paper, “CTCF-Mediated Human 3D Genome Architecture Reveals Chromatin Topology for Transcription,” the PacBio data set complemented short-read sequence data. Lead authors Zhonghui Tang, Oscar Junhong Luo, and Xingwang Li, along with senior author Yijun Ruan, worked with collaborators at the Jackson Lab and several other institutes to better understand genome organization and its implications for transcription.
The scientists used ChIA-PET (a much-needed abbreviation for “chromatin interaction analysis by paired-end tag sequencing”) to perform 3D mapping of the genome in four types of human cells, focusing on interactions mediated by transcription catalyst RNAPII and CTCF, which is known to play a role in genome folding. Separately, they studied gene expression with long-read sequencing.
“We find that CTCF/cohesin-mediated interaction anchors serve as structural foci for spatial organization of constitutive genes concordant with CTCF-motif orientation,” the authors report, “whereas RNAPII interacts within these structures by selectively drawing cell-type-specific genes toward CTCF foci for coordinated transcription.” They also show that changes in haplotype or allele interactions affect chromosomal configuration and alter gene expression.