PacBio Blog

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.

The influenza virus is known to develop rapid resistance to antiviral medications, but the extremely short infection cycle — less than two weeks in an average healthy host — has historically made it difficult to pinpoint evolutionary mutations as they arise. The team used SMRT® Sequencing, performed by the Icahn Institute at Mount Sinai, to analyze the H3N2 viral genome and reconstruct haplotypes, providing a clear phylogenetic view of the virus evolving over time within the host.

Among the findings, the scientists report that “individual resistance mutations appeared weeks before they became dominant, evolved independently on co-circulating lineages, led to a genome-wide reduction in genetic diversity through a selective sweep, and were placed into new combinations by reassortment.”

Based on the long-read data, the scientists were able to reconstruct haplotypes for several large segments of the viral genome to learn more about gene reassortment in persistent influenza infection. This allowed the team to track viral lineage dynamics, observing minor lineages becoming dominant over time, particularly as drug-resistant variants emerged. The authors note that even though reassortment was an important driver in H3N2 evolution, “it may not always be frequent enough to break all patterns of segment linkage.” This conclusion was based on phylogenetic evidence demonstrating these linkage patterns, particularly between hemagglutinin- and matrix-encoding genome segments. These association matches have also been observed across the general population, according to the scientists.

Rogers et al. hope this work will contribute to the ability to predict the emergence of drug-resistant influenza strains and to the development of antiviral therapies that prevent or reduce the occurrence of resistance. They note, however, that the evolutionary processes they documented must be studied in more detail. “In particular, a better understanding of segment linkage and reassortment, and whether they differ between mammalian and avian influenza viruses, may enable more accurate predictions of the rapidity with which particular genomic combinations can be obtained, including those mediating drug resistance, antigenic escape, and host adaptation,” they conclude.

Want to know more about viral sequencing using PacBio® technology? Check out these additional resources:

Human immunodeficiency virus (HIV) sequencing

Characterization of adeno-associated virus (AAV) genomic integration sites

De novo assembly of Pseudorabies virus

Human papilloma virus (HPV) sequencing

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.

Thursday, June 11, 2015

Updated! Data Release: Human MCF-7 Transcriptome


Our R&D team has added a new dataset for the MCF-7 human breast cancer transcriptome, originally released in 2013. The new results were produced using 28 SMRT® Cells with 4-hour movies and P5-C3 chemistry. Sizing was performed with the SageELF™ platform (fractions collected: 1-2 kb, 2-3 kb, 3-5 kb, and 5-10 kb). Sequencing of the larger fractions with our newer sequencing chemistry that generates longer reads added longer transcripts (up to 10 kb) to the MCF-7 dataset, which previously had only transcripts up to 4 kb.

New FASTA and GFF files are available, representing the new combined dataset. Raw data for both the 2013 and 2015 sequencing is also available.

Tuesday, June 9, 2015

Attend Our Worldwide User Meetings & SMRT Informatics Developers Conference

If you’d like to hear about the latest applications of SMRT® Sequencing from users, we have several events coming up. Our worldwide user group meetings and workshops feature PacBio users sharing their latest research, tips, and protocols, as well as our staff providing training and updates on products and methods to optimize your research. We’re always humbled by the quality and variety of science presented at these meetings. And for the bioinformatics crowd, we have a new event in August focused on developing new analytical tools for PacBio® data.

Tuesday, June 2, 2015

In Assembler Evaluation, Scientists Recommend Non-hybrid Approach to Bacterial Genomes

A new publication in Nature Scientific Reports recommends using only the PacBio® system to sequence bacterial genomes for the best chance of generating an accurate and finished assembly.

The paper, “Completing bacterial genome assemblies: strategy and performance comparisons,” reviews several different long-read assembly methods for bacterial genomes. Authors Yu-Chieh Liao, Shu-Hung Lin, and Hsin-Hung Lin from the Institute of Population Health Sciences in Taiwan note that while several methods exist, efforts to evaluate and compare them have been insufficient. They set out to thoroughly assess these methods, which include hybrid assembly protocols as well as long-read-only protocols.