PacBio Blog

Tuesday, September 30, 2014

New Papers Detail Complexity of Methylome-Related Virulence in Human Pathogens

In two new publications, one published today, scientists from Australia, Italy, the UK, and the US report critical and surprising new findings about DNA methylation-related complexity of bacteria. Adding to the list of advances from genome-wide epigenetic analysis, these projects enhance our understanding of how methylation systems work in human pathogens — and offer important clues for future investigations into how to treat them.

Today’s paper, “A random six-phase switch regulates pneumococcal virulence via global epigenetic changes,” was published in Nature Communications by scientists at the University of Leicester, University of Siena, University of Adelaide, and Griffith University. Senior authors Marco Oggioni and Michael Jennings and their collaborators studied Streptococcus pneumoniae, a bacterium responsible for serious infectious diseases including pneumonia, to figure out how the organism shifts between relatively benign and highly pathogenic phases.

To analyze the pathogen’s methylome, the scientists used Single Molecule, Real-Time (SMRT®) Sequencing. They found six different biological phases of the organism, each associated with a different level of virulence and characterized by a distinctive methylation pattern. Perhaps most importantly, they also found a genetic switch that enables S. pneumoniae cells to be randomly assigned to one of these six phases. “We show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system,” the authors write. They posit that the system is a key regulatory element designed to help the organism adapt to different host niches.

Now that scientists have determined the methylation profiles with the PacBio® platform, it should be possible for other scientists to accurately assign the pathogen to its specific phase. “Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo,” the authors note. “We believe these findings represent a new paradigm in gene regulation in bacteria and therefore are of great significance to the infectious disease field.”

Marco Oggioni said that dealing with S. pneumoniae’s genetic switch “is like being simultaneously confronted with six different bacteria; it gives them an unfair advantage, but knowing the genetic basis now places us in an optimal position to reinvestigate drug and vaccine efficacy.”

A separate paper from scientists at Griffith University and the Research Institute at Nationwide Children’s Hospital also looks at phase variation in a bacterium. “ModM DNA methyltransferase methylome analysis reveals a potential role for Moraxella catarrhalis phasevarions in otitis media” came out in The FASEB Journal earlier this month.

In it, senior author Kate Seib and her collaborators describe using SMRT Sequencing to characterize the methylome of Moraxella catarrhalis, a bacterium associated with childhood ear infections and complications of chronic obstructive pulmonary disease. The analysis revealed critical information about pathogenicity and its link to a phase-variable methyltransferase. Follow-up proteomic studies suggest that the phasevarion regulates expression in genes linked to infection, colonization, and defense against host organisms. “The modulation of gene expression via the ModM [phasevarion], and the significant association of the modM3 allele with otitis media, suggests a key role for ModM phasevarions in the pathogenesis of this organism,” the authors report.

Tuesday, September 23, 2014

Science Perspective: “Tracking Antibiotic Resistance”

In the current issue of Science there is an interesting Perspective by Scott Beatson and Mark Walker of the University of Queensland discussing research published this week in Science Translational Medicine by Conlan et al. who used SMRT® Sequencing to track plasmid diversity of hospital-associated infectious bacteria at the NIH Clinical Center.

The article provides a nice overview of the paper, including an explanation of the important role that plasmids play in spreading antibiotic resistance. They illustrate why short-read DNA sequencing technologies are insufficient in resolving them and long reads are necessary for this work.

“Plasmids may be viewed as the ‘dark matter’ of short-read bacterial genome assemblies, with many large-scale genomic studies conspicuously avoiding the complexities of plasmid structure. Genomic comparisons such as that described by Conlan et al. reveal how the dynamism in the structure and arrangement of resistance elements can only be realized by ‘closing’ plasmid genomes with long-read sequencing,” they write.

Even Sanger sequencing can be too difficult, time-consuming, and expensive for these types of projects, especially when dealing with multiple plasmids, they explain.

And while cataloging the antibiotic resistant genes in a particular bacterium may be the easy part, determining how these genes fit within plasmids is more difficult. With long-read sequencing you get a “complete picture” of the plasmid, “including the number, position, and context within mobile elements of every acquired antibiotic-resistance gene,” they note.

The authors conclude by saying, “Long-read genome assembly offers clear advantages for the resolution of complete plasmid sequences that can discriminate plasmid diversity, antimicrobial-resistance gene context, and multiplicity. Such information will enhance our understanding of plasmid carriage, transfer, epidemiology, and evolution.”

You can also read our blog post on the Conlan et al. paper.

Monday, September 22, 2014

Maryland Scientists Produce High-Quality, Cost-Effective Genome Assembly of Loa loa Roundworm Using SMRT Sequencing

A paper just released in BMC Genomics details what authors call “the most complete filarial
nematode assembly published thus far at a fraction of the cost of previous efforts.” The project was performed using the PacBio® RS II DNA Sequencing System by scientists at the University of Maryland School of Medicine’s Institute for Genome Sciences and the Laboratory of Parasitic Diseases at the National Institute of Allergy and Infectious Diseases.

In this genome sequencing effort, scientists generated a de novo assembly of Loa loa, a roundworm that infects humans. L. loa, transmitted to humans by deer flies, causes loiasis. The parasite lives under the skin and can grow to several centimeters without being detected.

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.

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.