PacBio Sequencing

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    PacBio Global Virtual User Meeting 2021
    Tuesday, November 9, 2021 @ 8am-2pm PST (Americas region)

    Featuring GC3F's own Research Assistant Maggie Weitzman
    on the Sample Prep Expert Panel 
    at 11:30am PST!

    Highly accurate long-read sequencing, or HiFi sequencing, is being used around the world to inspire discoveries in human biomedical research, plant and animal sciences, and microbiology and infectious disease. This year, the Global Virtual User Meeting will showcase research using PacBio powered technology from whole genome sequencing for de novo assembly to comprehensive variant detection to RNA sequencing and more!

    Register now for a 24-hour virtual journey (with 3 sessions for each global region) to join fellow researchers and hear from experts to learn how HiFi sequencing is expanding knowledge of genomes and inspiring advancements in science. On the agenda page, simply filter by the region of your choice, or this agenda link is pre-filtered for the Americas session (8am-2pm PST).

    ​​​​​Hope to see you there!

    If you want to get started on your own PacBio project, contact GC3F at genomics@uoregon.edu to discuss project details, and then it's as easy as creating an iLab  account to submit your samples!

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    GC3F IS NOW A CERTIFIED SERVICE PROVIDER (CSP)
    FOR PACBIO SEQUEL II SERVICES !!!

    Here at GC3F, we continue to expand our capabilities in long-read PacBio Single Molecule, Real-Time (SMRT) sequencing for generating high-quality genome assemblies, comprehensive transcriptome analyses, structural variant analyses, and epigenetic information.

    As a proven provider of PacBio services since 2017, and now a Certified Service Provider starting in 2021, we offer the Sequel II System for affordable, high-throughput studies of microbes, plants, animals, and humans.

    PacBio SMRT technology consistently produces some of the longest average read lengths available in the industry. These long read lengths combined with high consensus accuracy (up to 99.99% = Q40 -- or better!), uniform coverage, and simultaneous epigenetic detection means that SMRT Sequencing delivers valuable insights that previously have been unavailable to the scientific community.

    GC3F has achieved the PacBio Certified Service Provider status by undergoing a rigorous validation and training process. The certification shows that our lab can deliver the highest-quality services for SMRT Sequencing platforms.

    Contact us at genomics@uoregon.edu to get started on your next SMRT Sequencing project, and create your iLab account to submit your samples today!

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      GC3F ACCESS DURING COVID-19 PANDEMIC

      We are currently operating according to policies described in our approved Stage 1 Recovery Plan.

      A summary of these policies is posted up around the core, as well as HERE.

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      FAQs ON GC3F's OPERATIONAL STATUS DURING COVID-19 PANDEMIC

      CLICK HERE to get answers to our most frequently asked COVID-19 operational status questions.

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      NEW POLICY ON DATA RETENTION

      We have had an increasing number of requests for the retrieval of old sequencing data from our archive. While we are always happy to help whenever we can, a couple of issues have arisen surrounding this. CLICK HERE to review our new policy on data retention & retrieval.

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      OUR PACBIO SEQUEL II INSTRUMENT IS HERE!

      With PacBio SMRT technology, Single Molecules of native DNA are sequenced in Real Time on the Sequel II instrument utilizing devices called "SMRT cells."  Curious about how this works?  Watch this.

      Thanks to updated sequencing chemistry, the Sequel II instrument produces reads of improved length and quality compared to the previous Sequel instrument, and also boasts 8x higher throughput (8M ZMWs per cell vs. 1M previously). The Sequel II can be used for all your favorite sequencing applications:

      • Long-insert genomic (CLR = Continuous Long Read) libraries for de novo assembly, structural variant detection, etc.
      • HiFi genomic (CCS = Circular Consensus Sequencing) libraries for highly accurate reads for de novo assembly, SNP calling, small variant detection, etc.
      • Multiplexed microbial libraries
      • IsoSeq (transcript isoform) libraries
      • Small insert libraries constructed from amplicons, plasmid digests, capture pulldowns, etc.

       

       

      AND, compared to Sequel I, the new Sequell II produces an
      8-fold increase in read number per SMRT Cell 8M,
      for only ~40% higher cost than the previous SMRT Cell 1M!

      In fact, the new Sequel II actually produces data at a
      lower cost per Gb than our Illumina HiSeq 4000!

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      Some other benefits of switching to the Sequel II (compared to Sequel I):

      • We can obtain 8x the data from a single SMRT Cell 8M in the same amount of time to run 8 successive SMRT Cells 1M, which drastically reduces the sequencing run time and gets you your data faster.
      • We can now tack on an extra 10 hours movie time per SMRT cell (up to 30 hrs total).
      • For long-insert genomic CLR libraries, this means you can get higher coverage of your ultra-long insert molecules, which can improve your assembly quality.
      • For mid- and short-insert libraries, this means you can get even more read passes (subreads) of each molecule, giving you even higher CCS accuracy.
        • CCS accuracy of >Q20 can be achieved with ~4 subreads per molecule
        • CCS accuracy of >Q30 can be achieved with ~8 subreads per molecule
        • Note that CCS accuracy is a trade-off with insert length, i.e. longer inserts will receive fewer subreads and thus have lower CCS accuracy.

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      Here are some ESTIMATED RUN OUTPUTS to help you plan your next Sequel II project using v2/v2.1 sequencing chemistry

      Please treat these numbers strictly as ESTIMATES, not guarantees. Due to high performance variability between different libraries, we cannot guarantee a specific output! However, if you do plan to submit several projects to us, this would allow us to optimize loading conditions for your library type on successive runs.

      • Total # of raw reads, per SMRT Cell 8M = Target 4-5.5M reads

      • Total Gbases of raw data output (pre-CCS) per SMRT Cell 8M = 

        • Estimated 100-150 Gb for long-insert genomic CLR libraries (single or multiplexed)
        • Estimated 200-500 Gb for mid-insert genomic HiFi libraries. Note: After performing CCS analysis on this genomic HiFi data, PacBio's yield estimate is 20 Gb of HiFi data >Q20 per cell.
        • Estimated 150-400 Gb for short-insert libraries (IsoSeq, amplicons, plasmid digests, capture pulldowns, etc.)
           
      • Movie time per cell = 0.5 to 30 hours
         
      • Pre-extension time per cell = 0 to 12 hours (typically 0-4 hrs is needed)

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      CLICK HERE to submit a request for PacBio library prep, sequencing, or HMW gDNA extraction!

      (Note, you will need to register for an iLab account.)

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      SUBMISSION GUIDELINES FOR USER-PREPPED LIBRARIES:

      If you are planning to prep your own PacBio libraries, you'll just need to submit the PacBio Sequencing request in iLab. Please register for an iLab account and then click "Request Services" from the top menu bar in iLab. It is important that your Sequencing request has been submitted in iLab before you provide the physical samples to us!

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      SUBMISSION GUIDELINES FOR INPUT RNA/DNA FOR GC3F TO PREP YOUR LIBRARIES ON SITE:

      If you are planning to provide GC3F with RNA/DNA for us to prep the PacBio libraries for you, you'll need to submit BOTH the Sample Prep and the PacBio Sequencing requests in iLab. (If you'd like us to extract your samples as well, an additional Sample Prep request is required for that.) Please register for an iLab account and then click "Request Services" from the top menu bar in iLab. It is important that all of your requests have been submitted in iLab before you provide the physical samples to us!

      The quality of the input material is extremely critical for ensuring adequate library yield and high sequencing quality. Please be sure your purified dsDNA meets the following strict PacBio requirements:

      • Tissues have preferably been flash-frozen in liquid nitrogen and stored immediately at -80°C (other collection/storage methods may also be acceptable)
         
      • All steps of extraction, purification, and storage are performed in Eppendorf LoBind tubes
      • Submit only dsDNA (ssDNA cannot be prepped!)
        • If you are requesting IsoSeq prep, please contact us to discuss options for your RNA
      • Quantify your dsDNA using a fluorometric assay such as Qubit or Quant-iT (spectrophotometric assays are not adequate)
        • Be sure to very thoroughly (but very gently!) homegenize your sample using wide-bore tips & tap- or flick-mixing prior to taking an aliquot for quantification. If your gDNA is ultra HMW or very highly concentrated, it may require additional homogenization methods prior to quant (e.g., tube rotator or gentle thermomixer, extended time spent at room temp or 37°C, etc.)
      • Submit DNA that is of high quality = 
        • Fragments are of sufficient length for your desired sequencing application (contact us for details on your specific project)
        • No RNA present
        • Has not been exposed to intercalating dyes, UV radiation, ethidium bromide, high temperatures, extreme pHs, or vortexing
        • Minimal introduction of nicks, gaps, and other "undetectable" single-stranded damage
        • Minimal freeze-thaw cycles (also allow sample to fully thaw before any mixing, as ice chunks can shear the DNA)
        • If shipping to our facility, must be sent frozen on dry ice in a well-padded vessel to prevent tube cracking
      • Submit DNA that is highly purified =
        • Purified and stored in EB or TE buffer
        • 260/280 nanodrop ratio = at least 1.8
        • 260/230 nanodrop ratio = between 2.0 and 2.2
        • No visible particulates, precipitates, colors, or cloudiness
      • For genomic CLR libraries: Submit a minimum of 3 µg of gDNA per SMRT cell to be prepped, but 5-20 µg is preffered (more is always better and may allow us to "scale up" the prep reaction in order to select for the longest possible fragments)
        • If you have less than 3 µg of gDNA or are working with a small genome, please contact us to discuss options
      • For genomic HiFi libraries: Submit a minimum of 5-10 µg of gDNA per SMRT cell to be prepped. Reaction "scale ups" are not applied as frequently for Hifi preps, since the fragment size is constricted to a narrow range. However, this higher 5-10 µg input may  be required for just a "single scale" reaction because the shearing & size selection can substantially drop yield. 
        • If you have less than 10 µg of gDNA or are working with a small genome, please contact us to discuss options
      • For all other library prep types: The minimum input required will vary depending on the prep method used. Please contact us to discuss options!

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      SHIPPING GUIDELINES:

      Please deliver your samples to Maggie Weitzman in 288 Klamath Hall if you are an on-campus user.  For off campus users, please mail samples (tissues and/or extracted nucleic acids) frozen on dry ice in a well-padded vessel to prevent tube cracking. Jostling of thawed samples during shipping can cause undetectable damage to your samples that can inhibit proper sequencing. We have also seen many times that unpadded tubes will CRACK during shipment!

      NOTE: Please ensure that your package will arrive for delivery at UO only on Mon-Thurs. Due to campus restrictions during the COVID-19 pandemic, UO cannot accept deliveries on Fri-Sun.

      University of Oregon/GC3F
      1318 Franklin Blvd
      Room 273 Onyx Bridge
      Eugene, OR 97403

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      Please contact Maggie Weitzman for further details on PacBio sample submission. Thank you!

       

      We do our best to give an accurate estimate of turnaround time when service requests are submitted. These estimates are not guarantees and occasionally change due to unforeseen circumstances (instrument maintenance/repairs, staff sick leave, etc.). Please let us know if you have a hard deadline on project completion when you submit your service request and we will take measures to ensure that we either meet that deadline or inform you ASAP if circumstances beyond our control will cause us to miss it. Thanks!