PacBio Blog

Friday, April 17, 2015

AACR 2015: A Novel Look at Cancer, and a New SMRT Sequencing Grant Program

We’re looking forward to the annual meeting of the American Association for Cancer Research, which kicks off this weekend in Philadelphia. From directly phasing variants to sequencing full-length gene isoforms and other complex events, many scientists are already using SMRT® Sequencing to make exciting discoveries in cancer research. We hear from customers that the single-molecule approach opens the door for experiments they could not have done any other way.

If you’ll be at AACR, we encourage you to attend the talk from UCSF’s Catherine Smith on Monday at 10:40 a.m. in room 201. Her presentation, “Polyclonal and heterogeneous resistance to targeted therapy in leukemia,” will report on studies of patients with acute myeloid leukemia or chronic myeloid leukemia using the PacBio® system. The team focused on compound mutations, or multiple mutations, often distant from each other, on the same allele. These mutations appear to be indicative of resistance to tyrosine kinase inhibitors, something Smith and her colleagues have explored extensively since their finding that FLT3 is a valid therapeutic target for some patients with leukemia.

There are also posters with data from PacBio studies. Don’t miss the one from scientists at the Icahn School of Medicine at Mount Sinai (abstract #2398) reporting the use of SMRT Sequencing with other technologies to detect and monitor circulating tumor DNA in patients with ovarian or endometrial cancer. In a poster from scientists at Harvard and PacBio (abstract #4898), learn how the use of full-length isoform sequencing in the MCF-7 cell line allowed for the detection of complex splicing events, including novel fusion transcripts in genes associated with breast cancer.

We’ll be kicking off our newest grant program at AACR, giving one deserving scientist free access to SMRT Sequencing for up to four samples. To enter, simply submit a 250-word proposal telling us how targeted long-read sequencing will drive discoveries in your cancer research. Deadline for submission is May 22.  You do not need to attend AACR to enter! Official rules are available here.

To learn more, please stop by booth #2057, where the PacBio team will be happy to help you determine how SMRT Sequencing could be applied to your research.

Wednesday, April 15, 2015

In Genome-wide Study, Long Reads Prove Critical for Structural Variant Discovery

In a paper just published in BMC Genomics, a team of scientists led by Baylor’s Human Genome Sequencing Center reports a thorough analysis of structural variation in a personal genome. What makes this study special is the large number of different technologies applied and the sheer volume of data gathered and analyzed for this single genome. The paper also includes the first known analysis of structural variation in a diploid human genome using SMRT® Sequencing, with 10x coverage from PacBio® long reads.

Lead authors Adam English and William Salerno and their collaborators at a number of institutions describe the results obtained from a structural variant calling tool they have developed called Parliament. (Check out the full paper: “Assessing structural variation in a personal genome—towards a human reference diploid genome.”)

Structural variants account for the majority of variable bases in a human genome, according to the authors, who note that it will be important to detect and characterize these elements to understand their clinical relevance. Despite their significance, these variants are not as well understood as single nucleotide variants and other small variants. Through this effort to establish new ways to find and analyze structural variants, the scientists determined that short-read sequencing technologies alone miss a good amount of this kind of variation in a genome.

Working with a well-characterized genome, the team combined array CGH data with genome sequence data from Illumina, SOLiD, and PacBio systems, as well as a genome map from BioNano Genomics for the most comprehensive data set possible. That information was fed into Parliament, a pipeline for consensus structural variant calling that can be used with multiple data sets and detection approaches. The data sets were analyzed in various permutations within Parliament, which identified more than 31,000 loci representing possible structural variants. Of those, nearly 10,000 — spanning almost 60 Mb and nearly 2% of the reference genome — were supported by deep-dive genome analysis.

Of the 9,777 confirmed structural variants, the authors report that 3,801 were identified solely by PacBio long-read sequence data, “indicating the importance of read length when characterizing structural variation.” English et al. make the case for using multiple data sources to improve structural variant detection. “The addition of long-read data can more than triple the number of [structural variants] detectable in a personal genome,” they write.

The team has made Parliament publicly available through cloud-based service provider DNAnexus. “Implementation of Parliament on local compute requires independent installation of multiple discovery tools and a local assembler, imposing a burden of systems administration and resource consumption,” English et al. write, explaining why they chose DNAnexus to handle the computational side of this project. The cloud provider is now hosting the workflow established by the team — including a pipeline that takes BAM files and generates structural variant calls — as well as data generated in this project.

The authors hope their work helps establish a gold-standard catalog of human structural variation. “The present work identifies upper (4.5%) and lower (1.8%) estimates of the extent of structural variation in a personal genome and characterizes the impact of various resequencing methods,” they write. They also note that “as with [single nucleotide variants], many [structural variants] in a personal genome represent rare or private variants not observed in databases,” highlighting the need to sequence many individuals to obtain a deeper understanding of the extent and diversity of structural variants in the human population and their link to disease.

Thursday, March 26, 2015

In Chronic Myeloid Leukemia Study, SMRT Sequencing Detects Resistance Mutations Early, New Splice Isoforms and More

Scientists from Uppsala University report in a recent paper that using the Iso-Seq™ method with SMRT® Sequencing allowed them to detect and monitor mutations in the BCR-ABL1 fusion gene for patients with chronic myeloid leukemia (CML). Screening mutations in this region is important for determining the point at which these patients become resistant to tyrosine kinase inhibitor (TKI) therapies, and is currently performed in the clinic using Sanger sequencing, quantitative RT-PCR, and other assays.

The paper, “Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing,” was published last month in BMC Cancer from lead author Lucia Cavelier and collaborators. In it, the scientists describe sequencing samples from six patients who experienced poor response to cancer treatment; samples were collected at diagnosis and at subsequent follow-up periods and sequenced on the PacBio® system.

Tuesday, March 3, 2015

AGBT Highlights, Day Three: Genomic Medicine, Population Specific Genomes, Goats & Influenza

Day 3 of the AGBT conference was packed with interesting talks - we've covered a few highlights below.  Admittedly, it took a little more caffeine than usual to power through the day.....

In the clinical session, Euan Ashley from Stanford told attendees that genomic medicine is no longer something that we’re aiming for; it’s already here and being used routinely. He expressed concerns about accurate mapping of short-read sequence data for clinical utility, adding that the community needs to make progress in understanding complex genomic regions. Ashley noted that we still don’t have a gold-quality human genome with every single base known, and that achieving that remains an important goal for the field.

Friday, February 27, 2015

AGBT 2015: PacBio Workshop Review & Recording

Our AGBT workshop attracted more than 500 attendees thanks to the high-profile speakers who shared their perspectives on human genomic research. Because of the exclusivity of AGBT, we decided to live-stream our workshop to reach the broader scientific community. Thanks to the the hundreds of people who tuned in to our live webcast from afar! Here are some highlights from the presentations and the recording of the workshop is at the bottom of this post.

AGBT Highlights, Day Two: Human Genomes, Variation, and the Rapidly Evolving Y Chromosome

The first full day of AGBT kicked off with a great talk from Evan Eichler from the University of Washington. Starting with the premise that characterizing genetic variation is key to understanding phenotypes, his presentation offered in-depth looks into human genome projects designed to fully represent data missed in existing assemblies and current whole genome sequencing studies. Eichler pointed out that short-read sequencing misses a lot of structural variation, particularly when it occurs near repeat-rich regions. He said that every genome sequenced with short-read technology is missing important variation, and that a big problem is our inability to quantify just how much is missing. Eichler told attendees that he uses SMRT® Sequencing because it allows direct observation of native DNA, offers long reads, and has very little GC bias. He presented two sequencing projects focused on hydatidiform moles (CHM1 and CHM13), which have haploid human genomes. In one project, he reported detecting 26,015 structural variants, and closing or shrinking of 90 gaps in the human reference genome (many of which included GC-rich sequence) and adding a total of 1.1 Mb of novel sequence. He noted that one of the most important findings of the work was that 92 percent of insertions and 60 percent of deletions found in the genome were novel — including many in protein-coding regions — perhaps indicating how much has been missed in previous human genome population studies. An analysis of STRs found in the SMRT Sequencing-generated assembly showed that they were 3x more abundant and 2.8x longer than STRs in the existing human reference genome, which also suggests that current knowledge is incomplete. (Much of this work was included in this Nature paper from Chaisson et al.)

Thursday, February 26, 2015

AGBT Highlights, Day One: Advancing Human Reference Assembly & Sequencing in the Clinic

It is great to be here in Marco Island for the AGBT meeting! The 16th annual meeting hit the ground running with a pre-meeting workshop hosted by the Genome Reference Consortium (GRC) followed by an opening session that was more clinically focused than many attendees are used to at this tech-heavy conference. From the dynamic Q&A sessions, it was clear that these were precisely the kind of talks that people have been looking for as this meeting evolves downstream along with genomic science.

The GRC workshop, entitled ‘Advancing the Human Reference Assembly’ included four speakers: Valerie Schneider (NCBI), Tina Graves-Lindsay (TGI), Karyn Meltz Steinberg (TGI) and Deanna Church (Personalis, Inc.)  They stated that the current human genome reference assembly represents a mixture of over 70 individuals’ genomes in a single linear sequence. Thanks to population sequencing efforts, like the 1,000 Genome Project, we now know that there are regions of the human genome that are highly polymorphic, with multiple haplotypes that are segregating in the global population. Many of these regions (like MHC and KIR) are heavily associated with disease and immune-response.

Tuesday, February 24, 2015

AGBT 2015: Seeing the Genome in a New Light (Sunshine?)

Like many others, we’re looking forward to an exciting week of science and sun at the 16th annual Advances in Genome Biology and Technology (AGBT) conference! We’re hosting a lunch workshop on Friday, February 27, in the Palms Ballroom from 12:00 pm to 2:00 pm EST. We hope you can join us onsite (please reserve your seat) and even if you’re not at the conference, you can watch the live stream.

Wednesday, February 4, 2015

High-Quality Genome Assembly and Transcriptome of Cotton Using SMRT Sequencing

A recent research partnership with KeyGene, a Dutch plant genomics and crop improvement company, has resulted in an integrated whole-genome assembly and transcriptome of Gossypium hirsutum, or tetraploid cotton. This is the first known complete assembly for a polyploid crop with a genome larger than 2 Gb.

KeyGene has a long established reputation for generating high-quality data even for very complex genomes. For this project, the cotton genome was sequenced with 38x coverage using Single-Molecule, Real-Time (SMRT®) Sequencing. Assembly of PacBio® long reads reduced the number of contigs from more than 1 million in an existing short-read assembly to fewer than 22,000, representing a 47-fold increase in contiguity.

Thursday, January 29, 2015

Register Now: Isoform Sequencing Webinars Offer Tips on Method and Analysis

If full-length transcript information would be useful for your research, please join us for two upcoming webinars. Our scientists will offer tips for how to optimize the Iso-Seq™ method with the PacBio® System to meet your research goals.