Cost-effective method accurately orders DNA sequencing along entire chromosomes

This graphic art piece shows  the 23 pairs of chromosomes in 

humans. Image: Jane Ades, NHGRI

A new computational method has been shown to quickly assign, order and orient DNA sequencing information along entire chromosomes.  The method may help overcome a major obstacle that has delayed progress in designing rapid, low-cost — but still accurate — ways to assemble genomes from scratch.  Data gleaned through this new method can also validate certain types of chromosomal abnormalities in cancer, research findings indicate.

The advance was reported Nov. 3 in Nature Biotechnology by several University of Washington scientists led by Dr. Jay Shendure, associate professor of genome Sciences.

Existing technologies can quickly produce billions of “short reads” of segments of DNA at very low cost.  Various approaches are currently used to put the pieces together to see how DNA segments line up to form larger stretches of the genetic code.

However, current methods produce a highly fragmented genome assembly, lacking long-range information about what sequences are near what other sequences, making further biological analysis difficult.

“Genome science has remained remarkably distant from routinely assembling genomes to the standards set by the Human Genome Project,” said the researchers.  They noted that the Human Genome Project tapped into many different techniques to achieve its end result.  Many of these are too expensive, technically difficult, and impractical for large-scale initiatives such as the Genome 10K Project, which aims to sequence and assemble the genomes of 10,000 vertebrate species.

Members of the Shendure lab that developed what they hope will be a more scalable strategy were Joshua N. Burton, Andrew Adey, Rupali P. Patwardhan, Ruolan Qiu, and Jacob O. Kitzman.

To more completely assemble genomes, they tapped into a technology called Hi-C, which measures the three-dimensional architecture and physical territories of chromosomes within the nuclei of cells. Hi-C maps the physical interactions between regions of the chromosomes in a genome, including contact within a chromosome and with other chromosomes.  The results indicate which regions tend to occur near each other within three-dimensional space in a cell’s nucleus.

The researchers speculated that this interaction data, because it offers clues about the position of and distances between various regions of the chromosome, might reveal how DNA sequences are grouped and lined up along entire chromosomes.   They wondered if the interaction data could show them which regions of the genome are near each other on each chromosome.

Their investigation of this possibility led them to create what they named LACHESIS (an acronym for “ligating adjacent chromatin enables scaffolding in situ”), and also the Fate that measures the thread of destiny.

The map of physical interactions generated by Hi-C was interpreted by the LACHESIS computational program to assign, order and orient genomic sequences into their correct position along chromosomes, including DNA positioned close to the centromere, the “pinch waist” gap in the chromosome shape.

The researchers combined their new approach with other cheap and widely used sequencing methods to generate chromosome-scale assemblies of the human, mouse and fruit fly genomes. The researchers were able to cluster nearly all scaffolds — collections of short DNA segments whose position relative to each other is unknown — into groups that corresponded to individual chromosomes.

They then ordered and oriented the scaffolds assigned to each chromosome group, and validated their results by comparing them to the high-quality reference genomes for these species that were generated by the Human Genome Project. In the case of human genome, they achieved 98 percent accuracy in assigning tens of thousands of sequences of contiguous DNA to chromosome groups and 99 percent accuracy in ordering and orienting these sequences within chromosome groups.

“We think the method may fundamentally change how we approach the assembly of new genomes with next-generation sequencing technologies,” noted Shendure.

While he and his team cite many areas in which the computational and experimental methods can be improved, the approach is an important step in his lab’s long-term goal to facilitate the assembly, for a variety of species, of low-cost, high-quality genomes that meet the rigorous standards set by the Human Genome Project.

The research was supported by grants HG006283 and T32HG000035 from the National Human Genome Research Institute, and graduate research fellowships from the National Science Foundation.

Source: http://www.washington.edu/news/2013/11/07/cost-effective-method-accurately-orders-dna-sequencing-along-entire-chromosomes/

Largest, most accurate list of RNA editing sites

Researchers have compiled the largest and most rigorously validated list to date of genetic sites in fruit flies where the RNA transcribed from DNA is then edited by an enzyme to affect a wide variety of fundamental biological functions. The list yielded several biological insights and can aid further research on RNA transcription because flies are a common model in that work.

A research team centered at Brown University has compiled the largest and most stringently validated list of RNA editing sites in the fruit fly Drosophila melanogaster, a stalwart of biological research. Their research, which yielded several insights into the model organism’s fundamental biology, appears Sept. 29 inNature Structural & Molecular Biology.

The “master list” totals 3,581 sites in which the enzyme ADAR might swap an “A” nucleotide for a “G” in an RNA molecule. Such a seemingly small tweak means a lot because it changes how genetic instructions in DNA are put into action in the fly body, affecting many fundamental functions including proper neural and gender development. In humans, perturbed RNA editing has been strongly implicated in the diseases ALS and Acardi-Gutieres disease.

The new list of editing sites could therefore help thousands of researchers studying the RNA molecules that are transcribed from DNA, the so-called “transcriptome,” by providing reliable information about the thousands of editing changes that can occur.

“Drosophila serves as a model for all the organisms where people are studying transcriptomes,” said the paper’s corresponding author Robert Reenan, professor of biology in the Department of Molecular Biology, Cell Biology, and Biochemistry at Brown. “But in the early days of RNA editing research, the catalog of these sites was determined completely by chance – people working on genes of interest would discover a site. The number of sites grew slowly.”

In fact, Reenan was co-author of a paper in Science 10 years ago that made a splash with only 56 new editing sites which at the time, more than doubled the number of known sites in the entire field.

Validation means accuracy

Several more recent attempts to catalog RNA editing sites have yielded larger catalogs, but those contained many errors (the paper provides a comparison between the new list and previous efforts such as ModENCODE).

To avoid such mistakes, Reenan and colleagues, including lead author and graduate student Georges St. Laurent, painstakingly validated 1,799 of the sites. They worked with Charles Lawrence, professor of applied mathematics and the paper’s co-senior author, to predict another 1,782 sites and validated a statistically rigorous sampling of those.

In all, the team’s methodology allowed them to estimate that the combined list of 3,581 directly observed and predicted sites is 87 percent accurate.

“The sites that we validated, for anyone who wants to do the same experiment under the same conditions, the sites should be there,” said co-author and postdoctoral researcher Yiannis Savva. “In other papers, they just did sequencing to say there is an editing site there, but when you check, it’s not there.”

The researchers used the tried-and-true, decades-old Sanger method of sequencing to double-check all the candidate editing sites that they had found using the high-throughput technology called single molecule sequencing. They compared the sequenced RNA of a population of fruit flies to their sequenced DNA and to the RNA of another population of flies engineered to lack the ADAR editing enzyme. By comparing these three sequences they were able to see the A-to-G changes that could not be attributed to anomalies in DNA (i.e., mutations, or single-nucleotide polymorphisms) and that never occurred in flies incapable of editing.

As they conducted their validations, they fed the results back into their prediction algorithm. Over several iterations, that computer model “learned” to make better and better predictions. They ultimately found 77 different variables that helped them to distinguish real editing sites from nucleotides that were conclusively not editing sites.

Biological insights

The researchers then examined the implications of the patterns they saw in their data and gained several insights.

One was that a considerable amount of editing occurs in sections of RNA that do not code for making proteins. Editing is concentrated in a small number of RNAs, raising the question, Lawrence said, of what accounts for that selectivity.

“How does the cell go about choosing which ones are going to get edited and which aren’t is an interesting question this opens,” he said.

Where editing is found, the researchers discovered, there is usually more alternative splicing, which means the body is more often assembling a different recipe from its genetic instructions to make certain proteins.

The researchers also found that the RNAs that are most heavily edited tend to be expressed to a lesser extent, decreasing how often they are put into action in the body.

RNA editing helps explain why organisms are even more different from each other – and from themselves at different times — than DNA differences alone would suggest.

“RNA editing has emerged as a way to diversify not just the proteome but the transcriptome overall,” Reenan said.

In addition to Reenan, Lawrence, Savva, and St. Laurent, who is also affiliated with the St. Laurent Institute in Cambridge, Mass., the paper’s other authors are Michael Tackett, Sergey Nechkin, Dimitry Shtokalo, and Philipp Kapranov of the St. Lawernce Institute, Denis Antonets of the State Research Center of Virology and Biotechnology in Russia, and Rachael Maloney, a Brown graduate now at the University of Massachusetts Medical School.

Reenan received funding from the Ellison Medical Research Foundation.

Source: http://news.brown.edu/pressreleases/2013/09/rna

Precise mechanical manipulation of individual long DNA molecules

Electron micrographs demonstrating Aeon
Biowares' patented Molecular Threading technology.
Left: DNA molecules threaded onto an electron microscopy
grid with an amorphous carbon surface; right:
DNA molecules threaded onto a graphene
coated grid (credit: Aeon Biowares and KurzweilAI)

Teams of researchers from Harvard University and Halcyon Molecular, Inc. have disclosed “Molecular Threading,” the first technology to allow single DNA molecules to be drawn from solution and precisely manipulated, allowing for faster, cheaper, more accurate DNA sequencing.

This novel technology pulls single high-molecular weight DNA molecules from solution into air and then places them onto any surface. Halcyon Molecular developed the processes and the intellectual property is now owned by Palo Alto-based biotechnology firm Aeon Biowares.


“Molecular Threading offers a unique means of reaching from the macroscopic world into the world of large molecules with unprecedented exactitude,” says Dr. Chris Melville, CEO of Aeon Biowares and former Director of Chemistry at Halcyon Molecular. …

Additional details from the Aeon Biowares news release: “Molecular Threading, News of the First Public Disclosure“

Molecular Threading was invented by brothers Michael and William Andregg, the co-founders of Halcyon Molecular, Inc. Both co-authors on the current paper [open access: Molecular Threading: Mechanical Extraction, Stretching and Placement of DNA Molecules from a Liquid-Air Interface], the Andregg brothers were previously the subjects of a major profile in the national UK-based newspaper The Independent (“Silicon Valley: The anatomy of a cutting-edge start-up“, Sunday 14 August 2011).

The invention was spurred by the Andregg brothers’ quest for faster, cheaper, and more accurate DNA sequencing technologies. In particular, they needed a way to place DNA molecules onto surfaces in a more controlled manner than current techniques allow. As they tried many different techniques, including methods that are contrasted in the paper, they made an elegant discovery. A simple glass needle tip pulled from a Bunsen flame and coated with a hydrophobic polymer could stretch individual DNA molecules from water into air and place them onto a surface. Furthermore, due to the tension between the needle and the liquid, the DNA molecule is stretched in a geometrically predictable and reproducible way. The movie linked to here from the paper’s Supplementary Information shows how a bundle of DNA molecules remain normal to an air-water interface when stretched into air by the needle: DNA Thread Normal to Droplet Surface.

By attaching the needle to a piezo-positioner, they were soon able to make arrays of parallel stretched molecules as shown in the electron micrographs above. The image on the left shows DNA molecules threaded onto an electron microscopy grid with an amorphous carbon surface, while the image on the right shows DNA molecules threaded onto a graphene coated grid.

Molecular Threading is the enabling technology that allowed researchers for the first time to know the exact physical location of the DNA backbone. This together with the reproducible stretching meant that images that reveal the positions of the DNA bases could be used to determine the nucleotide sequence. This soon attracted the interest of Founders Fund partners Luke Nosek, Peter Thiel and Elon Musk, whose investments allowed the team to exploit Molecular Threading for DNA sequencing by electron microscopy.

Further characterization of the invention was performed by a collaboration of researchers working at Harvard University in the lab of George Church and by several researchers at Halcyon Molecular, some of whom are now working at Aeon Biowares. This includes Halcyon founding team member Kent Kemmish, now founder and CTO at Aeon Biowares.

“This is the first time anyone has ever pulled single high-molecule weight DNA molecules—or any macromolecules for that matter—out of solution and positioned them in a controlled way,” says Kemmish. “Though still in pre-commercial development, it is arguably one of the most advanced nanotechnologies in existence today.”

This advance is a very important nanotechnology that is especially important for DNA sequencing, and thus for personalized medicine, a major component of future medical technology. Much genomic DNA is riddled with many copies of repeated sequences. Current sequencing methods can only read a few hundred nucleotides at a time, so that determining the unique sequences flanking each copy of a repeated sequence can be very difficult. Precise manipulation of long DNA molecules opens the way to reading much longer sequences than can be done with current technology. The disclosure publication speculates “Applications beyond sequencing include nanofabrication, such as aperiodic templates for organic or inorganic materials using DNA as an organizing scaffold, or precision-patterned DNA nanowire arrays.” Organizing complex arrays of catalytic properties could be a step toward building complex molecular machine systems, perhaps as a step toward atomically precise manufacturing.

—James Lewis, PhD

Source: http://www.foresight.org/nanodot/?p=5805