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Pacific Biosciences of California, Inc. Message Board

paulieme60 10 posts  |  Last Activity: Oct 26, 2014 5:23 PM Member since: Feb 12, 2002
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  • Genome sequencing and assembly
    wAu genome sequencing was initially performed using the Illumina platform on gDNA
    extracted from whole adult files. However, the resulting assembly was fragmented in the
    regions of most interest, with scaffold positions uncertain. A second round of sequencing was
    therefore performed using the PacBio RS II system to obtain longer reads in an attempt to
    improve the assembly, using gDNA extracted from cultured cells rather whole adult files.
    The Illumina data was used to correct errors in the PacBio reads, which assembled into a
    single contig.
    The achievement of a single contig assembly shows that PacBio represents an extremely
    useful new sequencing platform for rapid generation of finished bacterial genome assemblies.
    Furthermore, the generation of this single contig from a very small amount of DNA
    (approximately 2 ng), containing a substantial amount of host DNA contamination (~40%),
    suggests that PacBio is well suited to use in cases where it is hard to obtain a large amount of
    gDNA, including obligate endosymbionts, like Wolbachia, that cannot be cultured outside of
    host cells. The sequence generated was largely consistent with data produced using the
    Illumina platform, with only one single nucleotide polymorphism (SNP) between the two
    datasets. There were 88 indels relative to Illumina data; these were mostly single nucleotide,Conclusions
    In this study, a methodology for conveniently extracting Wolbachia gDNA for genome
    sequencing using an infected cell line has been successfully employed, and the PacBio RS II
    sequencing platform has proved a very useful tool for achieving a complete bacterial
    assembly, particularly when combined with Illumina sequencing. Using this approach, a
    single contig assembly has been generated for the genome of the wAu strain,(For link go to IHUB PACB M B)

  • paulieme60 paulieme60 Oct 11, 2014 10:15 PM Flag

    Found a link to the sequencing 99 Ebola virus genomes .(go to IHUB M.B.) ("The team sequenced 99 Ebola virus genomes from 78 people in Sierra Leone, who were diagnosed with Ebola in late May and mid-June. Sadly, five of over 50 co-authors of the paper lost their lives to Ebola virus before the paper was published.") !!

  • paulieme60 paulieme60 Oct 11, 2014 9:11 PM Flag

    After Release of 20 New Genomes, 100K Pathogen Project Now Kicking PacBio Sequencing into Higher Gear
    July 30, 2013
    By Molika Ashford
    ... "He said PacBio sequencing has been "fantastic" for the team so far, yielding "nice
    .... reports on the sequencing of 99 Ebola virus genomes from infected patients in
    .... Get back to me when there's mobile sequence analysis."
    (sorry article from genomwb)To access $$$

  • Friday, October 10, 2014-- ASHG 2014: A New Look at the Human Genome with Long-Read Sequencing
    Scientists around the world are getting ready for the annual meeting of the American Society for Human Genetics taking place October 18-22 at the San Diego Convention Center. We’re looking forward to a number of excellent presentations and posters, and are delighted to see that many of them will focus on applying Single Molecule, Real-Time (SMRT®) Sequencing to human studies.
    (for link go to PACB website,click on blogs)

  • On short-read versus long-read sequencing
    Short-read sequencing technologies still maintain the advantage in terms of throughput, says Schadt, but there are a variety of important genomic features that cannot be characterized without long-read sequencing, such as long tandem repeats, bigger structural variations, and focal variants important in cancer.

    “I definitely think [short-read] technologies were tuned for certain problems and had certain advantages that enabled this big advance, but they are absolutely not hitting the entire problem like we need it hit,” he told Mendelspod.

    Cancer is a main area of study for which Schadt believes long-read sequencing is needed, in order to understand the complicated genomic features driving the tumor cells. And outside of human applications he called out plant genomics. “Plant genomes are so complicated and so flooded with repeat sequences, their only hope is to have long-read data,” he said.

    The quality of PacBio sequencing is 'beyond compare'
    Schadt noted that early misconceptions about the type of error profiles seen in single-molecule data erroneously led people to believe the data was of lower quality. He explained how the errors are random and can easily be washed out with a modest amount of coverage, whereas other next-generation sequencing technologies have systemic errors that cannot be removed.

    At Mount Sinai they used PacBio® technology to sequence the human genome and saw “very dramatic improvements in the quality of the de novo assemblies, revealing features that have never been seen before.” He said he believes this type of sequencing will become the standard. “The quality of that PacBio data is just beyond compare.”
    For full story (go to PACB website,click on blogs)

  • Reply to

    PacBio is ready to handle the challenge

    by paulieme60 Oct 2, 2014 3:36 PM
    paulieme60 paulieme60 Oct 2, 2014 3:41 PM Flag

    This is the best PART of the article."PacBio data is “really high quality” and “as good or better than Illumina and Sanger,” (Go to PACB website,click on blogs)!!

  • The challenge with short reads Geraghty explained that sequencing fosmids with short-read technology is cumbersome when it comes to stitching together the reads. Data analysis and finishing “became a roadblock that the Illumina short-read technology wouldn’t let us get beyond,” he said, noting that the finishing process takes 30 minutes to an hour per fosmid, prohibitive for any modest-scale effort. Geraghty marveled that he has received 40 kb reads from PacBio – meaning a whole fosmid can be sequenced in one piece.
    PacBio is ready to handle the challenge
    Geraghty said that with recent technology improvements, PacBio data is “really high quality” and “as good or better than Illumina and Sanger,” noting that his group has compared all three technologies with the same sequences. “It opens up a whole new possibility,” he said, because previously “you simply weren’t getting all of the data. People were using statistics to impute missing data and so on, and it simply doesn’t work.”
    Should PacBio be used for all major sequencing projects?
    Geraghty thinks so, noting that a resource such as the 1000 Genomes Project would be upgraded significantly with PacBio data for complex regions such as MHC and KIR. He said that if you look at these regions in the 1000 Genomes data you will find “a mass of confusion” because those regions are highly repetitive and contain a large amount of copy number and allelic variation, making it difficult or impossible to assemble the data correctly with short reads.
    “Any large human genome sequencing projects just using short-read technology are not going to acquire usable data for these complex regions, it’s as simple as that,” he said. For complex regions, “you’ll need long-read data,” he said, “The long-read data will give you really what everybody has been after all along without realizing it. It will give you the phase and the detail on the polymorphism in these highly polymorphic regions.”
    The future is bright (PACBIO Blogs)

  • Breakthrough study discovers six changing faces of ‘global killer’ bacteria
    Issued by University of Leicester Press Office on 30 September 2014

    "Every ten seconds a human being dies from pneumococcus infection making it the leading cause of serious illness across the globe." The University of Adelaide and scientists from Pacific Biosciences, and has for the first time shown a genetic switch that allows this bacterium to randomly change its characteristics into six alternative states.
    "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.”

    (Go to PACB web,click on blogs)also IHUB for another link.

  • 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. A comparison of short-read sequence data, short- and long-read hybrid data, and long-read-only data found that PacBio data used on its own outperformed other assemblies that included short-read sequence. The final assembly was produced with HGAP2 and polished with Quiver. It includes 96.4 Mbp in 2,250 contigs and covers about 9% more of the genome than a previous draft assembly — in 85% fewer contigs and starting with 80% less DNA, the authors note.

    (for link go to IHUB PACB M.B.)

  • 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

PACB
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