Tuesday, June 16, 2015

Stratos Genomics Develops Disruptive Gene Sequencing Technology

Seattle–based, Stratos Genomics Inc, an innovator in molecular engineering and gene sequencing techniques, announced today the successful demonstration of nanopore sequencing with their proprietary expandable nucleotides (X-NTP™) utilizing a novel single molecule detection method.

This advance–single molecule detection of long DNA sequences–builds upon previous success in which longer 210 and 36 base template sequences were the foundation of earlier work by the company.

Stratos’ Sequencing by Expansion technology (SBX™) represents a new platform for a low cost and more rapid method for whole genome sequencing. In a nutshell, SBXTM “is an efficient, low-cost DNA preparation method that rescales a DNA target into a longer surrogate polymer,’’ according to the company’s recent press release.

This surrogate–referred to as an Xpandomer™–is a reproduction of sequence data using what is known as “high signal-to-noise reporters,” thus allowing rapid identification of specific base sequences. This approach allows for single molecule detection of long sequences using small, low cost, nanopore instruments, also allowing for alternative measurement approaches. Representing a major advance, Stratos’ X-NTP sequencing was developed using a proprietary DNA polymerase to incorporate the expandable nucleotides.

“One year ago, we set a challenging goal to sequence X-NTP based Xpandomers in a nanopore prior to June and we accomplished it,” said Mark Kokorois, Stratos Genomics President and Chief Scientific Officer. “With the fundamental processes in place, our focus is now on optimizing for commercial level performance,” added Kokoris.

Roche , in June 2014, made a key investment in Stratos Genomics embarking on a research collaboration to pursue additional development of the specific technology enabling a transition to single molecule sequencing of DNA fragments with the aid of protein nanopores.

Over the past year, Roche has been partnering with Stratos to develop low-cost and rapid preparation techniques for DNA Xpandomers™. Roche’s expertise in polymerase mutagenesis, design of protein nanopores, modified nucleotide chemistries and rare reagent manufacturing was invaluable to the joint effort.

“The milestone sequencing results are promising,” explained Vinod Makhijani, Vice President and Project Leader, Roche Sequencing Business Development. “While several technical challenges remain on the path towards commercial readiness, we’re optimistic about our ability to tackle them with the joint expertise and resources of the Roche-Stratos team.”

“Roche Ventures and Fisk Ventures both have exercised their right under last year’s Series B financing to purchase additional shares based on our milestone success. Roche will invest an additional $10 million and Fisk Ventures $5 million. This will complete our $30 million Series B funding,” said Allan Stephan, CEO, Stratos Genomics.

The aim of Stratos Genomics, according to Stephan, is to make SBX the preferred method of DNA sequencing by ultimately making the ‘Sequencing by Expansion’ (SBX™) method low cost, rapid and widely available. SBX represents ground-breaking technology, utilizing a single-molecule detection method that removes the limitations of current, potentially error-prone sequencing technologies, essentially by promoting allowing a low-cost, rapid alternative to whole genome sequencing.

There are some companies that have attempted single molecule sequencing in different variations, including Pacific Bio and Oxford Nanopore, explains Stephan. However, Stratos’ patented single molecule detection technology, remains unique in their field of competitors at this stage.

One of the most widespread techniques currently available for genome sequencing–known as sequencing by synthesis (SBS)- was developed by Ilumina, based in Menlo Park, California.

SBS relies on creation of a large “forest” of uniform DNA segments, “almost like a field of the exact same pieces of DNA you are going to be measuring”, explains Stephan. The process involves floating in individual nucleotides with fluorescent capability. When a specific nucleotide is incorporated, your get a burst of light that optics can detect. “It’s a serial process of flooding in these nucleotides and reading the optical signatures that comes out of it”–which can be cumbersome “and involve big machines” described Stephan.

In comparison– in single molecule detection—as Stephan explains, “you don’t have to rely on an enormous ‘forest’ to give you a signal, but instead are getting a signal from the individual expandable base–using an Xpandomer.”

The single molecule detection approach to sequencing clearly represents a potentially disruptive technology in gene sequencing, as Stephan outlines.

“It can enable low cost, rapid, more accurate and targeted and whole genome sequencing, dropping the price into a range that is accessible by essentially everyone,” he concludes.

Source: Forbes

Monday, June 8, 2015

Intelligent Bacteria for Detecting Disease

Another step forward has just been taken in the area of synthetic biology. Research teams from Inserm and CNRS (French National Centre for Scientific Research) Montpellier, in association with Montpellier Regional University Hospital and Stanford University, have transformed bacteria into “secret agents” that can give warning of a disease based solely on the presence of characteristic molecules in the urine or blood. To perform this feat, the researchers inserted the equivalent of a computer programme into the DNA of the bacterial cells. The bacteria thus programmed detect the abnormal presence of glucose in the urine of diabetic patients. This work, published in the journal Science Translational Medicine, is the first step in the use of programmable cells for medical diagnosis.

Bacteria have a bad reputation, and are often considered to be our enemies, causing many diseases such as tuberculosis or cholera. However, they can also be allies, as witnessed by the growing numbers of research studies on our bacterial flora, or microbiota, which plays a key role in the working of the body. Since the advent of biotechnology, researchers have modified bacteria to produce therapeutic drugs or antibiotics. In this novel study, they have actually become a diagnostic tool.

Medical diagnosis is a major challenge for the early detection and subsequent monitoring of diseases. “In vitro” diagnosis is based on the presence in physiological fluids (blood and urine, for example) of molecules characteristic for a particular disease. Because of its noninvasiveness and ease of use, in vitro diagnosis is of great interest. However, in vitro tests are sometimes complex, and require sophisticated technologies that are often available only in hospitals.

This is where biological systems come into play. Living cells are real nano-machines that can detect and process many signals and respond to them. They are therefore obvious candidates for the development of powerful new diagnostic tests. However, they have to be provided with the appropriate “programme” for them to successfully accomplish the required tasks.

The transcriptor: the cornerstone of genetic programming

The transistor is the central component of modern electronic systems. It acts both as a switch and as a signal amplifier. In informatics, by combining several transistors, it is possible to construct “logic gates,” i.e. systems that respond to different signal combinations according to a predetermined logic. For example, a dual input “AND” logic gate will produce a signal only if two input signals are present. All calculations completed by the electronic instruments we use every day, such as smartphones, rely on the use of transistors and logic gates.

During his postdoctoral fellowship at Stanford University in the United States, Jérôme Bonnet invented a genetic transistor, the transcriptor.

The insertion of one or more transcriptors into bacteria transforms them into microscopic calculators. The electrical signals used in electronics are replaced by molecular signals that control gene expression. It is thus now possible to implant simple genetic “programmes” into living cells in response to different combinations of molecules[2].

In this new work, the teams led by Jérôme Bonnet (CBS, Inserm U1054, CNRS UMR5048, Montpellier University), Franck Molina (SysDiag, CNRS FRE 3690), in association with Professor Eric Renard (Montpellier Regional University Hospital) and Drew Endy (Stanford University), applied this new technology to the detection of disease signals in clinical samples.

Clinical samples are complex environments, in which it is difficult to detect signals. The authors used the transcriptor’s amplification abilities to detect disease markers, even if present in very small amounts. They also succeeded in storing the results of the test in the bacterial DNA for several months.

The cells thus acquire the ability to perform different functions based on the presence of several markers, opening the way to more accurate diagnostic tests that rely on detection of molecular “signatures” using different markers.
Principle of the use of modified bacteria for medical diagnosis. ©J. Bonnet/ Inserm.

“We have standardised our method, and confirmed the robustness of our synthetic bacterial systems in clinical samples. We have also developed a rapid technique for connecting the transcriptor to new detection systems. All this should make it easier to reuse our system,” says Alexis Courbet, a postgraduate student and first author of the article.

As a proof of concept, the authors connected the genetic transistor to a bacterial system that responds to glucose, and detected the abnormal presence of glucose in the urine of diabetic patients.

“We have deposited the genetic components used in this work in the public domain to allow their unrestricted reuse by other public or private researchers,[3]” says Jérôme Bonnet.

“Our work is presently focused on the engineering of artificial genetic systems that can be modified on demand to detect different molecular disease markers,” he adds. In future, this work might also be applied to engineering the microbial flora in order to treat various diseases, especially intestinal diseases.

This work received financial support from Inserm, CNRS, the Stanford-France Center for Interdisciplinary Studies, and Stanford University. Jérôme Bonnet is a recipient of the Atip-Avenir programme award, and is supported by the Bettencourt-Schueller Foundation.

[1] aimed at the rational engineering of artificial biological systems and functions
[2] (Bonnet et al. Science, 2013).
[3] available on: https://biobricks.org/bpa/

Source: Inserm