How is DNA Sequencing Used in Cancer Therapy?


Cancer therapy is increasingly aimed at the fundamental abnormalities within cancer cells – the genes and proteins that normally keep cell division under control, but are damaged or faulty in tumor cells. To understand which genes are abnormal, where they’re located within the genome, and how they affect cell growth, doctors and scientists use a procedure called DNA sequencing.

Sequencing DNA involves determining the precise order of the four chemical building blocks, or “bases,” that make up the DNA molecule. The bases – designated A, C, T, and G for the first letters of their chemical names ­– spell out the genetic code for each cell and each organism. Human cells have about six billion bases in all, arranged in pairs along the entire length of DNA. That figure is roughly equal to six gigabytes of data, or the number of letters in a book with three million pages.

Knowing the sequence of bases – also called nucleotides – helps scientists understand how different segments of DNA function. Some segments contain genes, which are blueprints for proteins produced by the cell. Other segments – more numerous and occupying vastly more space than genes – control whether genes are switched on or off. That is, they determine whether genes are actively being “read” by the cell to produce proteins, or are merely “on file” in the nucleus. Other regions of DNA have no known function and may be holdovers from evolutionary wrong turns and detours.

DNA sequencing explores the fundamental abnormalities within cancer cells.

Since DNA constitutes the “operating manual” of cells, errors in the arrangement of bases can cause a cell to malfunction in a variety of ways.  Nowhere is that more evident than in cancer, the disease most associated with wayward genes. The kind of sequencing errors that crop up in cancer cells can take several forms. These include mutations, in which one base is incorrectly swapped for another; copy number alterations, in which a gene or section of gene is repeated over and over or is missing altogether; and translocations, in which a stretch of DNA becomes stranded in the wrong part of the genome.

How is DNA Sequencing Used in Cancer Care?

Not every genetic misspelling or abnormality results in cancer. Many have no discernible effect on a cell’s function. But certain abnormalities are hallmarks of certain kinds of cancer. Doctors know, for example, that many non-small cell lung cancers (NSCLCs) have mutations in the gene EGFR. Targeted drugs are available that counter the effect of some of those mutations. By sequencing tumor cells from patients with NSCLC, therefore, doctors can often identify patients who are likely to benefit from those drugs.

Although the cost of DNA sequencing has dropped dramatically in recent years, it often isn’t feasible or affordable, or even necessary, to sequence all 3 billion base pairs within tumor cells. That’s why much of the sequencing done today is “whole exome sequencing,” which involves reading the bases only in sections of DNA that code for proteins.

At Dana-Farber, sequencing is the centerpiece of the Profile program, in which patients’ tumor tissue is scanned for hundreds of mutations or other abnormalities linked to cancer. The results of these scans can indicate patients who are good candidates for targeted therapies or for clinical trials in which potential new therapies are being tested.

DNA sequencing can also be used to identify people who may be at risk for certain types of inherited cancers. Scanning normal cells for mutations in the genes BRCA1 or BRCA2, for example, can indicate whether an individual has an above-average chance of developing cancers associated with those mutations. If so, there are a variety of measures such individuals can take to reduce their risk.

Sequencing and Cancer Research

Sequencing plays a large role in cancer research as well. Projects such as The Cancer Genome Atlas are sequencing thousands of tumor tissue samples to help uncover which genetic irregularities drive the growth of various types of cancer. Sequencing can also help researchers track how cancers change their genomic stripes over time. By sequencing the DNA in a tumor before and after treatment, for example, researchers hope to learn how cancer adapts to treatment and potentially becomes resistant to it.

An example of this use of sequencing is the PCROWD study at the Center for Prevention of Progression of Blood Cancers at Dana-Farber. Researchers are collecting tissue samples from people with precursor blood conditions, which develop into hematological cancers such as multiple myeloma and Waldenström macroglobulinemia, as well as other blood disorders. By genetically sequencing the cells in these samples, researchers hope to understand how these disorders evolve and to develop targeted drugs able to stop this progression in its tracks.

Circulating tumour DNA helps detect EGFR mutations


While biopsy remains the gold standard for EGFR mutation testing in patients with advanced non-small-cell lung cancer (NSCLC), circulating tumour-derived DNA (ctDNA) may provide a more feasible methodology, according to studies presented at the European Lung Cancer Conference (ELCC) 2015 held in Geneva, Switzerland.

“We were looking for a valid test that can identify an EGFR mutation when the tumour is not accessible for bronchoscopy or CT-guided biopsy, and that’s in agreement with the gold standard tissue test,” said Dr. Martin Reck from the Lung Clinic Grosshansdorf, Germany.

Reck reported data from the real-world ASSESS study, which compared tumour biopsy with plasma ctDNA in 1,162 matching samples from European and Japanese patients. “Mutation status showed a high 89 percent concordance between the two methods,” he said. “The sensitivity of the plasma test was 46 percent, specificity was 97 percent, and positive predictive value [PPV] 78 percent.” [ELCC 2015, abstract 35O_PR]

He noted that use of a highly sensitive DNA sequencing methodology and identical methods for tissue and plasma testing in a subset of patients further increased sensitivity to 72 percent, specificity to 99 percent and PPV to 94 percent.

“While improvements are still required in mutation analysis practices of both tissue/cytology and plasma samples, our data show that plasma ctDNA may be a feasible, suitable sample for EGFR mutation analysis,” he suggested. “It is important to use robust and sensitive methodologies to ensure patients receive appropriate treatment to address the molecular features of their disease.”

Another study reported the extraction of urine ctDNA to test for EGFR T790M mutation — a hallmark of disease progression in advanced NSCLC that is useful for patient monitoring. [ELCC 2015, abstract 36O]

The investigators obtained urine samples from patients who progressed on erlotinib and were confirmed to have EGFR T790M mutation by a tumour biopsy test. “EGFR T790M status was analyzed by a sensitive assay that had a lower limit of detection of 2 copies in a background of 20,000 copies of wild-type DNA,” explained Dr. Hatim Husain from the University of California, San Diego, CA, US.

Using this assay, they detected T790M mutation in 10 out of 10 confirmed EGFR T790M-positive patients (sensitivity, 100 percent). In addition, three patients with negative tissue testing results tested positive by urine analysis. EGFR T790M mutation was detected as early as 3.5 months prior to radiographic progression on first-line EGFR tyrosine kinase inhibitor (TKI) therapy, identifying five patients who may be eligible for second-line EGFR TKI treatment due to emergence of T790M mutation.

“This method, combining the extraction of urine ctDNA with an ultra-sensitive next-generation sequencing and mutation enrichment technology, has the advantage of urine as ctDNA source, potentially enabling dynamic monitoring of EGFR TKI therapy response from a completely noninvasive sample,” concluded Husain.

Whole Genome Sequencing And Its Implications: Can We Know Too Much?


DNA sequencing

Individuals who get their DNA sequenced can end up with a few clues to a diagnosis — but are mostly left with “500 various things of unknown significance.” 

What would you do if you had information about your entire genome — which told you the varying levels of risk you might have for diseases you’ve never heard of? Perhaps your genes would tell you that you had a high risk of developing Alzheimer’s disease, and suddenly your world would be different: you’d find yourself panicking every time you experienced a memory lapse, wondering if the clutches of dementia were already closing in, and that it’d only be a matter of time before you were in diapers and the world you knew washed away.

At the 2014 Forbes Healthcare Summit in New York City, Forbes Senior Editor Matthew Herper spoke about his experience getting his DNA sequenced and how it impacted — or didn’t quite impact — his life moving forward. Along with physician and genomics expert Dr. Robert Green, Herper discussed how knowledge of miniscule gene details could influence a person’s thinking, fears, and entire lifestyle.

Herper discovered he had hereditary hemochromatosis, or too much iron in his blood, as well as “500 various things of unknown significance,” he said. Basically, he didn’t find out anything of much consequence — nothing that would make him drastically change his life, or fear for his or his children’s future. But a pregnant woman getting her DNA sequenced and discovering that her unborn baby had a 1 in 4 chance of having a devastating mutation might feel differently.

The genome is an organism’s full set of DNA, and human genomes have about 3 billion DNA base pairs. A quick, cheap, and effective way of getting DNA sequenced would allow individuals to have access to a wide array of information that could then be used to improve their outcomes for certain diseases in the future, and spur them to improve their lifestyles (for example, people whose genes show they have a predisposition to cancer or diabetes might want to take control of their exercise and diet once they’re enlightened with information). This is where personalized medicine comes in — when a physician tailors treatments and approaches to a patient based on their unique genetic makeup. It could give people the power to inform themselves, and better themselves, Green said during the discussion.

The Burden Of Information

With all these clinical benefits aside, however, experts still wonder what knowing all of your DNA information really means for the individual. During the summit, Herper and Green discussed the implications of knowing too much — particularly among ordinary people who will eventually be taking advantage of the commercialization, and cheaper costs, of DNA sequencing in the future (the cost of getting your DNA sequence has decreased over the years, from hundreds of millions of dollars to anywhere between $1,000 and $10,000).

“What’s the burden of carrying around information that may not always manifest?” the speakers mused, referring to the fact that DNA sequencing can often claim a person has a slightly higher risk than others for a damaging hereditary condition, but that condition may not always manifest itself — nothing is certain with these tests. It’s the difference between people who are aware versus those who are unaware: ignorance is bliss, after all, and perhaps remaining unaware would be better for your mental health.

One audience member noted that sociological studies showed that people who are labeled hypertensive actually end up having higher hypertension than people who haven’t been labelled. That study, published in 2010, noted that “hypertension labeling might evoke feeling of fear of the affliction of a serious disease in a patient,” which “has been proven accurate by numerous investigators who have documented the negative psychological consequences of hypertension labeling.” It’s possible that the somewhat meaningless information a DNA sequence can often provide could have a greater psychological and emotional impact than we previously realized.

In a 2013 study, researchers wrote that though whole genome sequencing (WGS) was a valuable tool for disease diagnosis and improving treatments, it “raises difficult questions regarding what to do with the vast amounts of information generated that is incidental to the original purposes of sequencing, but have implications for health and well-being… Incidental information disclosure will be increasingly common as more and more physicians incorporate WGS into their practices, and it is likely to affect many patients’ health behaviors. The psychological processes behind changes… may be neither warranted nor beneficial.” In other words, learning about your genome could cause you some psychological distress even if it’s not warranted.

But Green, who is a physician-scientist at the Division of Genetics and the Department of Medicine at Harvard Medical School, believes that knowledge isn’t a burden, but rather is empowerment; and said that ordinary people should have access to their genomes as a matter of course in the future. The future is going to bring an increase in the use of whole genome sequencing, making it more available and accessible to the average consumer (some companies, like 23andme, have already attempted this). Ultimately, however, it depends on your personal choice: whether or not you believe that knowledge is power, or ignorance is bliss.

Researchers create unique graphene nanopores with optical antennas for DNA sequencing


High-speed reading of the genetic code should get a boost with the creation of the world’s first graphene nanopores – pores measuring approximately 2 nanometers in diameter – that feature a “built-in” optical antenna. Researchers with Berkeley Lab and the University of California (UC) Berkeley have invented a simple, one-step process for producing these nanopores in a graphene membrane using the photothermal properties of gold nanorods.

“With our integrated graphene nanopore with plasmonic optical , we can obtain direct optical DNA sequence detection,” says Luke Lee, the Arnold and Barbara Silverman Distinguished Professor at UC Berkeley.

Lee and Alex Zettl, a physicist who holds joint appointments with Berkeley Lab’s Materials Sciences Division and UC Berkeley’s Physics Department, were the leaders of a study in which a hot spot on a graphene membrane formed a nanopore with a self-integrated optical antenna. The hot spot was created by photon-to-heat conversion of a gold nanorod.

“We believe our approach opens new avenues for simultaneous electrical and optical nanopore DNA sequencing and for regulating DNA translocation,” says Zettl, who is also a member of the Kavli Energy Nanoscience Institute (Kavli ENSI).

Nanopore sequencing of DNA, in which DNA strands are threaded through nanoscale pores and read one letter at a time, has been touted for its ability to make DNA sequencing a faster and more routine procedure. Under today’s technology, the DNA letters are “read” by an electrical current passing through nanopores fabricated on a silicon chip. Trying to read electrical signals from DNA passing through thousands of nanopores at once, however, can result in major bottlenecks. Adding an optical component to this readout would help eliminate such bottlenecks.

Researchers create unique graphene nanopores with optical antennas for DNA sequencing
Luke Lee (left) and Alex Zettl led the creation of the world’s first graphene nanopores with a “built-in” optical antenna.

“Direct and enhanced optical signals are obtained at the junction of a nanopore and its optical antenna,” says Lee. “Simultaneously correlating this optical signal with the electrical signal from conventional nanopore sequencing provides an added dimension that would be an enormous advantage for high-throughput DNA readout.”

A key to the success of this effort is the single-step photothermal mechanism that enables the creation of graphene nanopores with self-aligned plasmonic optical antennas. The dimensions of the nanopores and the optical characteristics of the plasmonic antenna are tunable, with the antenna functioning as both optical signal transducer and enhancer. The atomically thin nature of the graphene membrane makes it ideal for high resolution, high throughput, single-molecule DNA sequencing. DNA molecules can be labeled with fluorescent dyes so that each base-pair fluoresces at a signature intensity as it passes through the junction of the nanopore and its optical antenna.

Read more at: http://phys.org/news/2014-11-unique-graphene-nanopores-optical-antennas.html#jCp

New material could enhance fast and accurate DNA sequencing


Gene-based personalized medicine has many possibilities for diagnosis and targeted therapy, but one big bottleneck: the expensive and time-consuming DNA-sequencing process.

Now, researchers at the University of Illinois at Urbana-Champaign have found that nanopores in the material molybdenum disulfide (MoS2) could sequence DNA more accurately, quickly and inexpensively than anything yet available.

“One of the big areas in science is to sequence the human genome for under $1,000, the ‘genome-at-home,'” said Narayana Aluru, a professor of mechanical science and engineering at the U. of I. who led the study. “There is now a hunt to find the right material. We’ve used MoS2 for other problems, and we thought, why don’t we try it and see how it does for DNA sequencing?”

As it turns out, MoS2 outperforms all other materials used for DNA sequencing – even graphene.

A nanopore is a very tiny hole drilled through a thin sheet of material. The pore is just big enough for a DNA molecule to thread through. An electric current drives the DNA through the nanopore, and the fluctuations in the current as the DNA passes through the pore tell the sequence of the DNA, since each of the four letters of the DNA alphabet – A, C, G and T – are slightly different in shape and size.

Most materials used for nanopore DNA sequencing have a sizable flaw: They are too thick. Even a thin sheet of most materials spans multiple links of the DNA chain, making it impossible to accurately determine the exact DNA sequence.

Graphene has become a popular alternative, since it is a sheet made of a single layer of carbon atoms – meaning only one base at a time goes through the nanopore. Unfortunately, graphene has its own set of problems, the biggest being that the DNA sticks to it. The DNA interacting with the graphene introduces a lot of noise that makes it hard to read the current, like a radio station marred by loud static.

MoS2 is also a single-layer sheet, thin enough that only one DNA letter at a time goes through the nanopore. In the study, the Illinois researchers found that DNA does not stick to MoS2, but threads through the pore cleanly and quickly. See an animation below:

“MoS2 is a competitor of graphene in terms of transistors, but we showed here a new functionality of this material by showing that it is capable of biosensing,” said graduate student Amir Barati Farimani, the first author of the paper.

Most exciting for the researchers, the simulations yielded four distinct signals corresponding to the bases in a double-stranded DNA molecule. Other systems have yielded two at best – A/T and C/G – which then require extensive computational analysis to attempt to distinguish A from T and C from G.

The key to the success of the complex MoS2 simulation and analysis was the Blue Waters supercomputer, located at the National Center for Supercomputing Applications at the U. of I.

“These are very detailed calculations,” said Aluru, who is also a part of the Beckman Institute for Advanced Science and Technology at the U. of I. “They really tell us the physics of the actual mechanisms, and why MoS2 is performing better than other materials. We have those insights now because of this work, which used Blue Waters extensively.”

Now, the researchers are exploring whether they can achieve even greater performance by coupling MoS2 with another material to form a low-cost, fast and accurate DNA sequencing device.

“The ultimate goal of this research is to make some kind of home-based or personal DNA sequencing device,” Barati Farimani said. “We are on the path to get there, by finding the technologies that can quickly, cheaply and accurately identify the human genome. Having a map of your DNA can help to prevent or detect diseases in the earliest stages of development. If everybody can cheaply sequence so they can know the map of their genetics, they can be much more alert to what goes on in their bodies.”

Secret Botulism Paper Published.


The discovery of a new form of the deadly botulinum toxin gets published, but its sequence is kept under wraps until an antidote is developed.

In a publishing first, the sequence of a newly discovered protein is not divulged in papers announcing the finding. Researchers at the California Department of Public Health in Sacramento discovered the protein, a new type of the extremely dangerous botulinum toxin, lurking in the feces of a child who displayed the symptoms of botulism. They published their findings in two reports on the website of The Journal of Infectious Diseases, but absent from either paper was the DNA sequence of the protein, the eighth form of botulinum toxin recovered from the bacteriumClostridium botulinum. The move represents the first time that a DNA sequence has been omitted from such a paper. “Because no antitoxins as yet have been developed to counteract the novel C. Botulinum toxin,” wrote editors at The Journal of Infectious Diseases, “the authors had detailed consultations with representatives from numerous appropriate US government agencies.”

These agencies, which included the Centers for Disease Control and Prevention and the Department of Homeland Security, approved publication of the papers so long as the gene sequence that codes for the new protein was left out. According to New Scientist, the sequence will be published as soon as antibodies are identified that effectively combat the toxin, which appears to be part of a whole new branch on the protein’s family tree.

The Fastest DNA Sequencer.


DNA sequencing has revolutionized medicine and biomedical research. For example, DNA analysis can tell doctors which drug might work best against a particular cancer. But current technology usually sequences only short stretches of DNA and can take hours or days.

To sequence anything longer than a few hundred base pairs, scientists mince up thousands of copies of the target DNA, sequence all the fragments, and use software to painstakingly reconstruct the order of the DNA bases by matching overlap within fragments. A new approach, called nanopore sequencing, can handle long strands of DNA at once, eliminating the need for overlap analysis. As a result, nanopore sequencers could be cheaper, faster, and more compact than other DNA sequencers. They can also accurately sequence stretches with many repeating base pairs. The MinION from Oxford Nanopore Technologies connects to a USB port. Soon, anyone with $1,000 and a computer will be able to sequence DNA.

Fastest DNA Sequencer Diagram
Davvi

1) Drop the DNA sample on a chip.
Researchers place pretreated samples—blood from a patient or purified DNA, for example—into a small port. Within the device is a silicon chip with many thin membranes studded with tiny pores.

2) Unzip the DNA.
An enzyme shuttles the DNA to the membrane’s nanopore. It then unzips the twin strands of DNA and feeds one end into the pore. The pore is a set of proteins arranged in a ring and derived from bacteria. The inner diameter of the pore is a couple of nanometers wide: 100,000 times thinner than a human hair.

3) Block the ion current.
Electrodes send an ionic current, a flow of ions, through the open nanopore. As a group of a few DNA bases—the As, Ts, Cs, and Gs—threads through the neck of the pore, it blocks the ions and interrupts the current. A sensor records the electrical disturbance.

4) Determine the sequence.
Software in an attached computer analyzes the electrical signal recorded for every group of bases. Because each combination of bases blocks the current in a distinctive fashion, the software can deduce the identity and sequence of the individual bases in the group. As the DNA strand feeds through the pore, the software stitches together the sequence of bases on the entire strand.

5) Check for errors.
The device can determine the sequence of a single strand of DNA, but for greater precision, it can also read the complementary strand. Once the first strand of the DNA ratchets through the pore, a small stretch of DNA called a hairpin structure acts as a tether to draw the matching half into the pore as well.

Treatable cancer subtype found.


Australian researchers have identified a potentially treatable subtype of pancreatic cancer, which accounts for about 2% of new cases. This subtype expresses high levels of the HER2 gene. HER2-amplified breast and gastric cancers are currently treated with Herceptin.

Pancreatic cancer is the fourth leading cause of cancer death in Western societies, with a 5-year survival rate of less than 5%. It is a molecularly diverse disease, meaning that each tumour will respond only to specific treatments that target its unique molecular make-up.

Sebastian_Kaulitzki_PancreaticCancer_shutterstock

A new study, published in Genome Medicine, used a combination of modern genetics and traditional pathology to estimate the prevalence of HER2-amplified pancreatic cancer. Pancreatic surgeon Professor Andrew Biankin, from Sydney’s Garvan Institute of Medical Research and the Wolfson Wohl Cancer Research Centre at the University of Glasgow, worked with pathologist Dr Angela Chou and bioinformatician Dr Mark Cowley from Garvan, as well as cancer genomics specialist Dr Nicola Waddell from the Queensland Centre for Medical Genomics at the University of Queensland.

Using data sourced from the Australian Pancreatic Cancer Genome Initiative1 (APGI), the team identified a patient with high-level HER2 amplification. Using whole genome DNA sequencing of the tumour, Dr Nicola Waddell pinpointed the specific region of the genome that contains HER2.

Dr Angela Chou then performed detailed histopathological characterisation of HER2 protein in tissue samples taken in the past from 469 pancreatic cancer patients. This produced a set of standardised laboratory testing guidelines for testing HER2 in pancreatic cancer, and showed the frequency of HER2 amplified pancreatic cancer of 2.1%. 

Dr Chou also found that – like HER2-amplified breast cancer patients – the cancers of those with HER2-amplification in the pancreas tended to spread to the brain and lung, rather than the norm, which is the liver.

Dr Mark Cowley analysed all the data generated by the project and compared it to other sequences from many cancer types produced by the International Cancer Genome Consortium and The Cancer Genome Atlas project. “HER2 amplification was prevalent at just over 2% frequency in 11 different cancers,” he observed.

“We make the case that if HER2 is such a strong molecular feature of several cancers, then perhaps recruiting patients to clinical trials on the basis of the molecular features rather than the anatomical region of their cancer could have a significant impact on patient outcomes, and still make economic sense for pharmaceutical companies.”

“Such ‘Basket trials’ as they are sometimes called, may advance treatment options for those with less common cancer types.”

In Australia, 2,000 people are diagnosed with pancreatic cancer each year, and so 40 are likely to have the HER2 amplified form. 

While Herceptin is available through the Pharmaceutical Benefits Scheme for treating breast and gastric cancer, it is not available for treating HER2-amplified pancreatic cancer as no clinical trial has yet been conducted to determine the drug’s efficacy in that case.

The Garvan Institute in collaboration with the Australasian Gastro-Intestinal Trials Group, is recruiting pancreatic cancer patients through the APGI for a pilot clinical trial, known as ‘IMPaCT’2, to test personalised medicine strategies. 

Potential patients will be screened for specific genetic characteristics, including high levels of HER2, based on their biological material sequenced as part of the APGI study. Once these characteristics are confirmed, patients will be randomised to receive standard therapy or a personalised therapy based on their unique genetic make-up.

 

Sequence-Based Discovery of Bradyrhizobium enterica in Cord Colitis Syndrome.


BACKGROUND

Immunosuppression is associated with a variety of idiopathic clinical syndromes that may have infectious causes. It has been hypothesized that the cord colitis syndrome, a complication of umbilical-cord hematopoietic stem-cell transplantation, is infectious in origin.

METHODS

We performed shotgun DNA sequencing on four archived, paraffin-embedded endoscopic colon-biopsy specimens obtained from two patients with cord colitis. Computational subtraction of human and known microbial sequences and assembly of residual sequences into a bacterial draft genome were performed. We used polymerase-chain-reaction (PCR) assays and fluorescence in situ hybridization to determine whether the corresponding bacterium was present in additional patients and controls.

RESULTS

DNA sequencing of the biopsy specimens revealed more than 2.5 million sequencing reads that did not match known organisms. These sequences were computationally assembled into a 7.65-Mb draft genome showing a high degree of homology with genomes of bacteria in the bradyrhizobium genus. The corresponding newly discovered bacterium was provisionally named Bradyrhizobium enterica. PCR identified B. enterica nucleotide sequences in biopsy specimens from all three additional patients with cord colitis whose samples were tested, whereas B. enterica sequences were absent in samples obtained from healthy controls and patients with colon cancer or graft-versus-host disease.

CONCLUSIONS

We assembled a novel bacterial draft genome from the direct sequencing of tissue specimens from patients with cord colitis. Association of these sequences with cord colitis suggests that B. enterica may be an opportunistic human pathogen.

Souirce: NEJM

 


Sequence-Based Discovery of Bradyrhizobium enterica in Cord Colitis Syndrome
BACKGROUND
Immunosuppression is associated with a variety of idiopathic clinical syndromes that may have infectious causes. It has been hypothesized that the cord colitis syndrome, a complication of umbilical-cord hematopoietic stem-cell transplantation, is infectious in origin.
METHODS
We performed shotgun DNA sequencing on four archived, paraffin-embedded endoscopic colon-biopsy specimens obtained from two patients with cord colitis. Computational subtraction of human and known microbial sequences and assembly of residual sequences into a bacterial draft genome were performed. We used polymerase-chain-reaction (PCR) assays and fluorescence in situ hybridization to determine whether the corresponding bacterium was present in additional patients and controls.
RESULTS
DNA sequencing of the biopsy specimens revealed more than 2.5 million sequencing reads that did not match known organisms. These sequences were computationally assembled into a 7.65-Mb draft genome showing a high degree of homology with genomes of bacteria in the bradyrhizobium genus. The corresponding newly discovered bacterium was provisionally named Bradyrhizobium enterica. PCR identified B. enterica nucleotide sequences in biopsy specimens from all three additional patients with cord colitis whose samples were tested, whereas B. enterica sequences were absent in samples obtained from healthy controls and patients with colon cancer or graft-versus-host disease.
CONCLUSIONS
We assembled a novel bacterial draft genome from the direct sequencing of tissue specimens from patients with cord colitis. Association of these sequences with cord colitis suggests that B. enterica may be an opportunistic human pathogen.
Source: NEJM

 

%d bloggers like this: