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Pacific Biosciences of California, Inc. Message Board

paulieme60 7 posts  |  Last Activity: Sep 18, 2014 5:01 PM Member since: Feb 12, 2002
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  • This class has been running since 2010-11, on the following principle:

    ¦autumn semester: isolate bacterial DNA, sequence using Illumina, assemble;
    ¦spring semester: close assembly gaps, annotate genome.
    As anyone following genomics knows, the times they are a’changing again and again, so this is less and less state-of-the-art. So we have decided to try a new course plan this year, taking advantage of the progress in bacterial genome sequencing with long PacBio reads.

    Our new principle is, hopefully:

    ¦autumn semester: isolate bacterial DNA, sequence using PacBio, assembly trivial, annotate genome;
    ¦spring semester: RNA-seq under 2 growth conditions, experiments, Illumina sequencing and bioinformatics analysis.
    “Hopefully” because PacBio on bacteria is not yet routine, depending on the genus and the growth conditions. We are thus trying two different bacteria, a Pseudomonas which has a cool story for the RNA-seq part, and a Caulobacter which has been shown to work with PacBio. Preliminary studies on the Pseudomonas are somewhat discouraging for the PacBio sequencing, but we will still try, with adaptations of the protocol. We will also keep the possibility to reverting to Illumina sequencing plus assembly, but we would like to avoid that (if Caulobacter is plan B, this is plan C).

    And of course, we have never done RNA-seq with master students, so this year will be a new adventure, comparable to our first course in 2010. Stressful and exciting.

    This entry was posted in course plan, sequencing. Bookmark the permalink.
    ? Our first student genome paper is out: Miyazaki et al Environ MicrobiolOne Response to A new adventure: PacBio sequencing and RNA-seq in the classroom
    Winship Herr says:
    September 18, 2014 at 14:31
    This is a super cool course. My first Master First-step project was to sequence 100 nucleotides of the lac operon

  • With Illumina, the dominant player in the NGS market, claiming this year that they’ve reached that target with their HiSeq X Ten system, it’s fair to stop and ask just what has been achieved. What do you get for that $1,000? And furthermore, where does NGS go from here?

    Beginning next week, we're launching a new series, The Rise of Long Read Sequencing.

    I first heard “long read” sequencing differentiated from “short read” in an interview with Mike Hunkapiller, CEO of Pacific Biosciences last year. I had asked him the obvious question about how he expects to compete with Illumina, and he responded saying that “short read technologies” had serious draw backs.

    “Wait a minute,” I remember thinking at the time, “did Mike just dismiss Illumina’s technology out right? And what are these long reads he’s talking about.”
    (Very interesting article)!! For link,Go to IHUB PACB M.B.

  • Illumina and Ion Torrent technologies have read lengths up to a few hundred base pairs, while Sanger sequencing covers several hundred. In contrast, Pacific Biosciences’ technology has average reads of about 8,500 bases. Some users have reached tens of thousands of bases. Its RS II system costs about $700,000.

    Pacific Biosciences’ single-molecule real-time sequencing is a sequencing-by-synthesis approach that doesn’t use an amplified set of DNA fragments and doesn’t require stopping and starting the reaction to add reagents and image results. Reactions on individual DNA molecules are tracked in real time across 150,000 nanoscale wells where isolated polymerases read the DNA and incorporate fluorescently tagged nucleotides. Because detection occurs only at the bottom of the wells, the background noise from the other reactions is reduced.

    Stability of the sequencing process depends in large part on the polymerase. Pacific Biosciences has modified a simple bacteriophage enzyme, slowing it down so that it incorporates about three bases per second and its detector can keep up. To prevent inadvertent photo damage that could stop the process, the company has put a protective scaffold on the enzyme.

    Although fast and cheap sequencing will yield much useful knowledge, it has come at a price because of the shorter read lengths, Korlach argues. Pacific Biosciences “wanted to build a technology first and foremost that gives the highest quality of sequence information,” he says.

    The 10-year-old company launched its first sequencer in 2011 and has since improved its chemistry, detection, and throughput. On target for 70% sales growth this year, to about $47 million, Pacific Biosciences has installed more than 100 systems and has a market share of a few percent. Its business has seen “a nice boost as the platform (For link go to IHUB)

  • Posted August 14, 2014-- New Results
    Assembling Large Genomes with Single-Molecule Sequencing and Locality Sensitive Hashing
    Konstantin Berlin, Sergey Koren, Chen-Shan Chin, James Drake, Jane M Landolin, Adam M Phillippy
    We report reference-grade de novo assemblies of four model organisms and the human genome from single-molecule, real-time (SMRT) sequencing. Long-read SMRT sequencing is routinely used to finish microbial genomes, but the available assembly methods have not scaled well to larger genomes. Here we introduce the MinHash Alignment Process (MHAP) for efficient overlapping of noisy, long reads using probabilistic, locality-sensitive hashing. Together with Celera Assembler, MHAP was used to reconstruct the genomes of Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, and human from high-coverage SMRT sequencing. The resulting assemblies include fully resolved chromosome arms and close persistent gaps in these important reference genomes, including heterochromatic and telomeric transition sequences. For D. melanogaster, MHAP achieved a 600-fold speedup relative to prior methods and a cloud computing cost of a few hundred dollars. These results demonstrate that single-molecule sequencing alone can produce near-complete eukaryotic genomes at modest cost.

  • The most recent version of this article [btu392] was published on 2014-07-17 (Published by Oxford University Press)---proovread: large-scale high accuracy PacBio correction through iterative short read consensus
    Motivation: Today, the base code of DNA is mostly determined through sequencing by synthesis as provided by the Illumina sequencers. Although highly accurate, resulting reads are short, making their analyses challenging. Recently, a new technology, Single Molecule Real-Time (SMRT) sequencing, was developed which could address these challenges as it generates reads of several thousand bases. But, their broad application has been hampered by a high error rate. Therefore, hybrid approaches which use high quality short reads to correct erroneous SMRT long reads have been developed. Still, current implementations have great demands on hardware, work only in well-defined computing infrastructures and reject a substantial amount of reads. This limits their usability considerably, especially in the case of large sequencing projects.
    Results: Here we present proovread, a hybrid correction pipeline for SMRT reads, which can be flexibly adapted on existing hardware and infrastructure from a laptop to a high performance computing cluster. On genomic and transcriptomic test cases covering Escherichia coli, Arabidopsis thaliana and human, proovread achieved accuracies up to 99:9% and outperformed the existing hybrid correction programs. Furthermore, proovread corrected sequences were longer and the throughput was higher. Thus, proovread combines the most accurate correction results with an excellent adaptability to the available hardware. It will therefore increase the applicability and value of SMRT sequencing.

  • FREE: Workshop, Lunch & Tour of Arizona Genomics Institute to see PacBio® RSII!!!
    Rare Opportunity - Register Now - Seating is Limited!

    Thursday, August 14th, 2014
    BIO5 Institute - Keating Bldg, Rm 103


    Sequencing with Long Reads: New and Upcoming Applications of PacBio® SMRT® Technology
    Jonas Korlach, Chief Scientific Officer, Pacific BioSciences

    Targeted PacBio® Sequencing: BAC Libraries, Physical Maps, Platinum Sequencing
    Rod A. Wing, Director, Arizona Genomic Institute (AGI)

    Microbial Genome Sequencing
    David Baltrus, Assistant Professor, School of Plant Sciences & Microbial Sciences

    Sequencing Large Eukaryotic Genomes
    Yeisco Yu, Sequencing Group Leader, Arizona Genomics Institute
    Dave Kudrna, BAC/EST Resource & Physical Mapping Center Group Leader

    Full-Length Transcript Sequencing with PacBio® Iso-Seq™ Method: Going Beyond Short Read Assembly
    Jonas Korlach, Chief Scientific Officer, Pacific BioSciences

  • Improving potato genetics using the long read sequencing data
    Expected/Ideal Start Date 01 Aug 2014
    The successful applicant will use and optimise tools for assembling a large plant gene sequences and an entire genome using ultra-long Pacific Bioscience (and as available Nanopore) reads, as well as using shorter Illumina reads to verify and correct the assembly.

    (for link go to IHUB PACB M B)

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