PacBio Blog

Wednesday, September 17, 2014

NIH Study: Finished Genomes Provide Actionable Data to Combat Spread of Drug-Resistant Bacteria

A study launched over concerns around hospital-acquired infections has led to a recommendation for better microbial screening of patients upon admission. The research, from scientists at several NIH institutes, found that cases of hospital-acquired infection were less common than cases where patients were likely already colonized but received false negative results from basic screening.

The study was made possible by Single Molecule, Real-Time (SMRT®) Sequencing, which allowed researchers to sequence plasmids and analyze their diversity and likely phylogeny. Short-read sequencing and strain-typing technologies could not provide the information necessary for a comprehensive analysis.

Single-molecule sequencing to track plasmid diversity of hospital-associated carbapenemase-producing Enterobacteriaceae,” published today in Science Translational Medicine, comes from senior author Julie Segre at the National Human Genome Research Institute. In the project, NIH scientists sequenced 20 isolates of carbapenem-resistant Enterobacteriaceae — including two from a Klebsiella pneumonia outbreak at the NIH Clinical Center in 2011, 16 from routine patient and environmental surveillance after the outbreak, and two from former NIH clinic patients found to be positive for the bacteria after being released.

According to the paper, carbapenem-resistant Enterobacteriaceae are a serious concern in healthcare because of their resistance to most or all antibiotics as well as their rapidly increasing incidence — they are now detected four times as often in patients as they were 10 years ago. Because these pathogens carry their drug resistance in plasmids, there is risk that resistance could be acquired by other bacteria. The scientists found it essential to develop a clear view of how these organisms functioned and spread among patients, but quickly determined that short-read sequencers couldn’t provide the whole picture. Plasmids were large — up to 200 kb — and full of repetitive and mobile elements that were intractable with short-read data.

SMRT Sequencing enabled the scientists to analyze and fully assemble all 20 genome sequences for various Enterobacteriaceae with carbapenem resistance. The genomes ranged from 3.9 Mb to 6.2 Mb, with the largest plasmid weighing in at 379 kb. The team independently validated the PacBio® data with optical mapping and short-read sequencing, finding that the accuracy for each genome was better than 99.9999% (Q60). After fully analyzing the genomes, the scientists confirmed that standard approaches such as PCR, multilocus sequence typing, and pulsed-field gel electrophoresis would not have been able to distinguish between certain strains, leaving out valuable information that would have helped track transmission.

Among the paper’s findings, Conlan et al. were able to trace the transmission path of the original carbapenem-resistant Klebsiella outbreak from its index patient to 17 other patients. They also found one patient who was colonized with two different strains of carbapenem-resistant Enterobacteriaceae, and a single patient who acquired the bacteria from another patient while in the hospital. “This case is the only example from the five suspected cases that resulted in a definitive conclusion that nosocomial transmission had occurred, again underscoring the power of genomic sequencing in the clinical setting,” the authors write. They were surprised to find just a few cases of horizontal plasmid transfer, noting instead that “the number of independent introductions observed was surprisingly high and indicated a complex network of plasmids with incredible diversity.” Finally, they identified a new plasmid encoding for carbapenemase that may have significant clinical impact.

“On the basis of our sequence data demonstrating that only 1 of the 10 cases represented nosocomial spread, we directed our resources toward surveillance at admission for carbapenemase-producing organisms,” the researchers report. The clinical team also increased measures aimed at containing bacterial transmission, including taking surveillance cultures from patients more often.

As antibiotic resistance becomes more common and new antibiotics remain rare, it is more important than ever to prevent infection, according to the authors. “In addition to implementing recommended infection control measures such as surveillance, hand hygiene, and barrier precautions, we must find more sophisticated methods to detect, track, and eradicate multidrug-resistant bacteria.”

Whole-genome sequencing is one powerful response that yields actionable medical data, they add. “Sequencing can elucidate the landscape of bacterial transmission, allowing hospitals to target funds and personnel for infection control interventions that provide the best patient care,” they write. “The cost of whole-genome sequencing is dwarfed by [other] costs associated with outbreaks and their investigations, including the human and financial toll and the loss of patient confidence in the health care facility.”

UPDATE: This paper came out at a pivotal time in the fight against antibiotic resistance. Just a day later, the White House issued a report from the President's Council of Advisors on Science and Technology entitled "Combating Antibiotic Resistance." At the same time, President Obama issued an executive order calling for the development of a national plan to address this challenge. Check out this blog post for a summary of the efforts.

Thursday, September 11, 2014

The Rise of Long Reads: Mendelspod Podcast Series

Mendelspod host Theral Timpson kicked off a new podcast series this week on long-read sequencing that will include interviews with luminaries in the genomics field. Check out this introductory article from Timpson for an explanation of why scientists are demanding longer reads to meet their research goals.

The first interview is with Mike Snyder at Stanford, who has published recent papers in Nature Biotechnology and PNAS using Single Molecule, Real-Time (SMRT®) Sequencing for transcriptome analysis and demonstrated that long reads enable full coverage of RNA molecules. He discusses that work and his views on long-read sequencing and transcriptomics on the show. Here are some highlights:

On the state of transcriptomics
Without using long-read sequencing, the way transcriptomes are figured out is “crazy,” Snyder explains. “We take RNA, we blow it up into little fragments, and then we try and assemble them back together to see what the transcriptome looked like in the first place. And that’s a horrible way to do this because what we’re really trying to do is understand all of the different isoforms of a transcript….So when you blow them up and try to reassemble them back together you can’t always figure out which parts of the puzzle belong together.” (This reminds us of a clever cartoon in Nature Methods last year, subscription required.)

Tuesday, September 9, 2014

Genome Analysis of Unicellular Organism Reveals Frequent, Massive Reshuffling

A recent publication from senior author Laura Landweber at Princeton University offers a remarkable and unexpected look at sweeping genomic rearrangements in a unicellular organism.

The Architecture of a Scrambled Genome Reveals Massive Levels of Genomic Rearrangement during Development,” published in Cell, comes from lead authors Xiao Chen and John Bracht as well as other collaborators from Princeton, the Icahn School of Medicine at Mount Sinai, Benaroya Research Institute, and other institutions.

Monday, August 18, 2014

Genome-Wide Methylation in Human Microbiome Samples

Scientists in Florida and Finland recently published a report of their work studying methylation patterns in two human microbiome samples. While microbiome studies have become quite popular, the authors note there have been no prior papers detailing genome-wide methylation of bacteria found in those studies. Their goal was to ascertain how much added functional variation might occur based on methylation patterns.

The methylome of the gut microbiome: disparate Dam methylation patterns in intestinal Bacteroides dorei,” published in Frontiers in Microbiology, comes from lead author Michael Leonard and senior author Eric Triplett at the University of Florida plus a team of collaborators from hospitals and universities across Finland.

Wednesday, August 6, 2014

Plant and Animal Genomes: New Web Resource Available

After so many compelling customer projects for microbial genomes, it’s been rewarding to see more scientists turning to Single Molecule, Real-Time (SMRT®) Sequencing for larger genomes, such as plants and animals. Many PacBio users are performing de novo sequencing and assembly or upgrading draft genomes initially generated by short-read technologies. Extraordinarily long reads and throughput improvements have allowed scientists to affordably assemble and close genomes such as the Atlantic cod, spinach, and Orpinomyces, an anaerobic fungus found in the rumen of cows, to name a few.

As reported by several customers at the 2014 Plant & Animal Genome conference in San Diego, new features of SMRT Sequencing, including the ability to identify full-length isoforms and automate haplotyping, are making it possible for researchers to generate high-quality, contiguous assemblies with improved genome annotations. A more holistic view offers scientists better insights into individual gene functions and their coordination within networks.

Tuesday, July 29, 2014

Novel Study of Genome-wide PT Modifications in Bacteria Performed with SMRT Sequencing

A recent paper from scientists in China and the United States demonstrates a novel view of phosphorothioate (PT) DNA modifications in two bacterial genomes. Scientists from Shanghai Jiao Tong University, Massachusetts Institute of Technology, Wuhan University, and Pacific Biosciences teamed up to deploy Single Molecule, Real-Time (SMRT®) Sequencing to generate the first genome-wide view of PT modifications and to better understand their function. “Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences” by Cao et al. was published in Nature Communications.

The authors note that PT modifications, which replace a non-bridging phosphate oxygen with sulphur, were only recently discovered to occur naturally in bacteria. (PT modifications are used by scientists to stabilize synthetic DNA molecules against nuclease degradation.) Today, these modifications have been seen in more than 200 bacteria and archaea, but the detailed genome-wide distribution and biological functions have not been clear.

Tuesday, July 22, 2014

At ISMB, Gene Myers’ Keynote Offers History, Future of Genome Assembly

At ISMB 2014 in Boston earlier this month, Gene Myers of the Max-Planck Institute for Molecular Cell Biology and Genetics, presented a keynote address entitled “DNA Assembly: Past, Present, and Future.”  Myers received the prestigious Senior Scientist Accomplishment Award from the International Society for Computational Biology (ISCB) at the event.

The ISCB Senior Scientist Accomplishment Award honors respected leaders in computational biology and bioinformatics for their significant contributions to these fields through research, education, and service. Myers is being honored as the 2014 winner for his outstanding contributions to the bioinformatics community, particularly for his work on sequence comparison algorithms, whole-genome shotgun sequencing methods, and for his recent endeavors in developing software and microscopic devices for bioimage informatics. 

Friday, July 11, 2014

ISMB 2014: The World Cup of Bioinformatics

We’re eager for the #ISMB conference — it’s the 22nd annual Intelligent Systems for Molecular Biology event — kicking off this weekend in Boston. As we continue to push our technology to deliver longer read lengths, we have been honored to work with many leading bioinformaticians to optimize the processing and analysis of our data.

Several of those experts will be speaking at ISMB this year. On Sunday, attendees will hear from Adam Phillippy of the National Biodefense Analysis and Countermeasures Center. He’ll be presenting at noon on producing complete genome assemblies using Single Molecule, Real-Time (SMRT®) Sequencing data. Adam’s team recently developed a new assembler called MHAP that dramatically reduces CPU power needed for building assemblies, so we are eager to hear more.

Wednesday, July 9, 2014

Optimizing Eukaryotic De Novo Genome Assembly: Webinar Recording Available
Our webinar on eukaryotic genome assembly attracted a great crowd, and now we’re making the full recording available to the community. The session featured great hands-on information and best practices for working with Single Molecule, Real-Time (SMRT®) Sequencing data. “Optimizing Eukaryotic Genome Assembly with Long-Read Sequencing” featured three excellent speakers — Michael Schatz and James Gurtowski from Cold Spring Harbor Laboratory and Sergey Koren from the National Biodefense Analysis and Countermeasures Center — and was hosted by our own CSO Jonas Korlach.

Schatz kicked off the session with an overview of assemblers for PacBio® data (as well as recommendations for when to use each one) and a look at the challenges of short-read assemblies. He also set expectations around long-read data, noting that for genomes less than 100 Mb, users should expect a nearly perfect assembly from the automated workflow. Genomes up to 1 Gb should be represented in a high-quality assembly with a contig N50 of at least 1 Mb. Genomes larger than that will have shorter contig N50 stats and will require larger computational power, he added.

Tuesday, July 1, 2014

Scientists Generate the First Personal Transcriptome Using SMRT Sequencing

A new paper from scientists at Stanford University and Yale University describes the use of Single Molecule, Real-Time (SMRT®) Sequencing to generate transcriptomes for three individuals. The work is believed to be the first personal transcriptome analysis using long-read sequencing.

The paper, entitled “Defining a personal, allele-specific, and single-molecule long-read transcriptome,” was published in PNAS by Hagen Tilgner, Fabian Grubert, Donald Sharon, and Michael Snyder. Last year, the same authors published a study using SMRT Sequencing to analyze transcriptomes across tissue samples from human organs. In the PNAS publication, they compare metrics from the new data set to those from the previous study.