PacBio Blog

Wednesday, August 26, 2015

In Bacterial Study, Scientists Link Epigenetic Switch to Virulence, Antibiotic Resistance, and More

Scientists from Griffith University, Ohio State University College of Medicine, and other institutions recently published a detailed study of phase-variable expression of a DNA methyltransferase in non-typeable Haemophilus influenzae, the predominant cause of pediatric middle ear infections. The team found that the bacterium’s epigenetic switch regulates proteins used in current vaccine candidates and influences important traits including antibiotic resistance, ability to evade the host immune system, and biofilm formation, which significantly contributes to chronic infection.

The paper, “A biphasic epigenetic switch controls immunoevasion, virulence and niche adaptation in non-typeable Haemophilus influenzae,” was published in Nature Communications last month by lead author John Atack, senior author Michael Jennings, and colleagues. Their study of H. influenzae used SMRT® Sequencing to elucidate the genome and epigenome of multiple bacterial strains collected across several decades. Closed genome assemblies were generated for all five bacterial strains sequenced.

The scientists aimed to identify the DNA recognition motifs for the bacterium’s five known alleles of the N6-adenine DNA methyltransferase, ModA, which has been linked to random on/off switching. Atack and colleagues found that these phasevarions actively alter gene expression in every strain analyzed, and also successfully identified a novel allele in this system. Based on SMRT Sequencing data, most alleles were classified as having “prototypical type III methyltransferase recognition sequences,” but one allele was found to recognize a four-base motif instead of the traditional five.

The impact of this allele-specific epigenetic switch on bacterial pathogenesis and disease was examined in detail. The scientists demonstrated that these phasevarions influence bacterial susceptibility to a number of antibiotics, including ampicillin and erythromycin, which are commonly used to treat H. influenzae infection. The group discovered that these switches influence the protein expression of several potential vaccine candidates currently in development. They also report that phasevarions are prevalent in H. influenzae strains found in healthy people, not just those associated with chronic infections.

The scientists also investigated the influence of these alleles using a chinchilla model for ear infection, providing the first in vivo evidence for consistent selection of the “on” phenotype for one of the phasevarion alleles. “This indicates that switching of the modA2 phasevarion plays an important role in niche adaptation to the middle ear,” Atack et al. write.

These results demand additional study, the authors say. “Defining the stable immunological target that NTHi [H. influenzae] represents requires a full analysis of the impact of phasevarions on NTHi gene expression, and future vaccine candidates will need to be assessed to confirm that their expression is not influenced by the epigenetic changes that result from phasevarion ON/OFF switching,” they write. “Bacterial epigenetics is a key emerging field in bacterial pathogenesis and a new challenge to vaccine development for these important human pathogens.”

Thursday, August 20, 2015

The Gapless Assembly: Scientists Describe Workflow for Producing Complete Eukaryote Genome

Sunflowers with verticillium wilt caused by V. dahliae
In a new mBio publication, scientists from Wageningen University and KeyGene in The Netherlands report results from several strategies used to assemble the genome of a filamentous fungus, and describe the specific pipeline they recommend for sequencing and assembling eukaryotic genomes.

Single-Molecule Real-Time Sequencing Combined with Optical Mapping Yields Completely Finished Fungal Genome” comes from lead authors Luigi Faino and Michael Seidl, senior author Bart Thomma, and collaborators. Using Verticillium dahliae as a model, which is a plant pathogen responsible for the damaging verticillium wilt disease in many crop species, they compared short-read and long-read sequencing approaches and incorporated optical mapping data to develop the method that generated the highest-quality assembly for the 36 Mb genome. This particular fungus was an ideal fit for the project, the authors note, due to its extensive genomic rearrangements and enrichment for repetitive elements.


Wednesday, August 12, 2015

Using SMRT Sequencing, Scientists Uncover Unexpected Transcript Diversity in Fungi

A new PLoS One publication from scientists at the Joint Genome Institute, University of Minnesota, and other organizations demonstrates that fungal genomes may contain far more transcript diversity than previously thought.

In “Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing,” lead author Sean Gordon, senior author Zhong Wang, and collaborators used long-read isoform sequencing to characterize four fungal species. In addition to widespread alternative splicing, they found evidence of polycistronic transcription units that could be important engineering targets for genetic manipulation of fungi.


Thursday, July 23, 2015

SMRT Sequencing Provides Novel View of Long-Term Viral Evolution in a Single Patient

A group of scientists from the University of Pittsburgh School of Medicine and New York University used long-read sequencing from PacBio for a remarkable new study characterizing influenza virus evolution with unprecedented precision.

Intrahost Dynamics of Antiviral Resistance in Influenza A Virus Reflect Complex Patterns of Segment Linkage, Reassortment, and Natural Selection,” published in mBio by lead author Matthew Rogers and senior author Elodie Ghedin, reports a two-year study tracking the flu virus in one person. Although normally limited to acute infection, in this case the patient, a three-year-old with severe combined immunodeficiency disease, received multiple antiviral therapies but kept shedding virus over the course of 21 months. The team was able to study 10 samples collected during that time period, using sequencing to track viral evolution in great detail. “This unique natural experiment provides a rare view into the patterns, dynamics, and mechanisms of drug resistance of influenza virus,” the team wrote.


Tuesday, July 21, 2015

Webinar: Anthony Nolan’s Neema Mayor Reports on HLA Typing with Long Reads

Following the recent paper about HLA typing from scientists at Anthony Nolan Research Institute, we thought readers might enjoy this webinar from Neema Mayor, a scientist at the institute and lead author on the paper. The video offers a great foundation on HLA typing for beginners as well as more detailed information about typing technologies for advanced users. (Learn more about the institute's plans for HLA typing in this GenomeWeb article.)

Named for Anthony Nolan, a young boy whose need for a bone marrow transplant spurred his mother to start the world’s first registry of potential donors, the institute focuses heavily on education and research, Mayor says. She and her colleagues brought in the PacBio® sequencing platform last year to assess the utility of long reads based on single molecules for HLA analysis, a process used for matching organs in transplant patients.


Tuesday, July 14, 2015

SMRT Sequencing Contributes to Detection of DNA Methylation in C. elegans

A recent paper in the journal Cell presents novel findings of DNA methylation in C. elegans, an organism previously believed not to have such epigenetic marks. Scientists used several approaches to analyze the adenine N6-methylation (6mA) found in C. elegans, including SMRT® Sequencing to directly observe base modifications across the genome.

From lead authors Eric Greer and Mario Blanco with senior author Yang Shi at Harvard Medical School, “DNA Methylation on N6-Adenine in C. elegans” describes a range of technological methods deployed to assess methylation across the worm’s genome. The team queried the nematode with specific antibodies for 6mA; immunofluorescence; ultra-high-performance liquid chromatography combined with triple-quadrupole tandem mass spectrometry; SMRT Sequencing; and MeDIPseq, an antibody-based immunoprecipitation paired with DNA sequencing.


Thursday, July 9, 2015

The Festival of Genomics Review: A Celebration of Long Reads

At the inaugural Festival of Genomics event in Boston, more than 1,500 people turned out to see what was billed as a conference unlike any other. The meeting was indeed unique, featuring a play (starring well-known scientists), a giant chess board, and a Genome Dome, in addition to the more familiar lineup of excellent speakers and workshops.

To help kick off the festival, genomic luminaries Craig Venter and James Lupski presented plenary talks on day 1 and set the stage for some exciting science to follow. Lupski’s talk was particularly impactful, as he described how his team at Baylor recently sequenced his own personal genome using 10-fold PacBio® long-read coverage to analyze copy number changes underlying his rare genomic disorder.


Monday, June 29, 2015

Nature Methods Paper Uses Long-Read Data for Highly Contiguous Diploid Human Genome

A new publication in Nature Methods describes a new single-molecule assembly approach that resulted in “the most contiguous clone-free human genome assembly to date,” according to lead authors Matthew Pendleton, Robert Sebra, Andy Pang, and Ajay Ummat.

The paper, “Assembly and Diploid Architecture of an Individual Human Genome via Single Molecule Technologies,” comes from a large team of collaborators at the Icahn School of Medicine at Mount Sinai, Cornell, Cold Spring Harbor Laboratory, and other institutions.


Thursday, June 25, 2015

SMRT Data Delivers for Next-Generation HLA Typing at Anthony Nolan Research Institute

A new publication in PLoS One from authors at Anthony Nolan’s Research Institute describes a feasibility study for HLA typing using SMRT® Sequencing. The research institute, where the world’s first bone marrow registry started in 1973, is part of the UK-based charity dedicated to improving the outcomes of bone marrow transplantation. Scientists at Anthony Nolan are leaders in HLA typing, which is an important step in matching a bone marrow or stem cell donor to a patient in need.

The Anthony Nolan team adopted the PacBio® system last year, and this publication reflects its efforts to test and establish the new standards for HLA typing. In "HLA Typing for the Next Generation," from lead author Dr Neema Mayor and senior author Professor Steven Marsh, they discuss the sequencing and analysis of various types of representative samples typically seen in their pipeline.


Monday, June 15, 2015

Scientists Publish New Methylation Analysis Protocols Using SMRT Sequencing

Scientists from the Icahn School of Medicine at Mount Sinai and the University of Saskatchewan teamed up to develop an innovative approach to methylation analysis using Single Molecule, Real-Time (SMRT®) Sequencing. The resulting method was just published in BMC Genomics.

Lead author Yao Yang and colleagues note in the paper [“Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS)”] that existing methods for methylation analysis are limited by cost and throughput in the case of Sanger sequencing, or short read lengths with NGS technologies. Their goal was to develop a method combining long reads, high accuracy, and high throughput.