PacBio Sequencing

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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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      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.

       

      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.)

       

      PacBio Sequencing Prices:

      Service

      Internal Rate

      External Rate

      Sequel II single SMRT Cell 8M sequencing $1,686 $2,360

       

      PacBio Sample Prep Prices:

      Service

      Internal Rate

      External Rate

      PacBio DNA sequencing library prep  $450 per sample $675 per sample
      PacBio Iso-seq RNA-seq library prep $200 per sample $300 per sample
      PacBio bacterial 16S amplicon library prep $3,000 per plate of 95 DNA samples $4,500 per plate of 95 DNA samples
      High molecular weight DNA extraction (1st round) $150 for 2 extractions from tissue $225 for 2 extractions from tissue
      High molecular weight DNA extraction (if 1st round yield is too low) $100 for each additional extraction $150 for each additional extraction

       

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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 gDNA from your samples as well, an additional Sample Prep request is required for extraction.) 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 PacBio library yield and high sequencing quality. Please be sure your purified nucleic acids meet the following strict QC (quality control) metrics:

       

      COLLECTION & STORAGE REQUIREMENTS

      • Freshly-harvested tissues have been flash-frozen in liquid nitrogen and immediately stored at -80°C until extraction (or shipping frozen on dry ice) is completed
        • Other collection/storage methods may also be acceptable, but are likely to result in lower sequencing quality
        • For plant extractions: Provide at least 3-5g of young leaves that have been dark-treated for 72 hours prior to flash-freezing
        • For other extractions: Amount of tissue required will vary substantially depending on your organism, tissue, and sequencing application. Please email genomics@uoregon.edu to discuss
           
      • All steps of extraction, purification, and storage are performed in Eppendorf LoBind tubes
         
      • Nucleic acids have been subjected to minimal freeze-thaw cycles (samples are also allowed to fully thaw before any mixing, as ice chunks can shear nucleic acids)
         
      • All shipped samples (tissues and/or nucleic acids) are shipped FROZEN on dry ice

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      QUALITY REQUIREMENTS

      • Submit only dsDNA for CLR/HiFi applications (ssDNA cannot be prepped!) or total RNA for Iso-Seq (post-RT/PCR cDNA may also be prepped into Iso-Seq libraries - contact us for details)

      • Fragments are of sufficient length for your desired sequencing application
        • For CLR/HiFi, it is strongly recommend to use samples with >50% of the gDNA >30kb in length
        • For Iso-Seq, it is strongly recommend to use samples with RIN/RQN >7
           
      • No contaminating nucleic acids present
        • For CLR/HiFi, samples have been treated with RNase and there is no remaining RNA present
        • For Iso-Seq, samples have been treated with DNase and there is no remaining DNA present
           
      • Samples have not been exposed to intercalating dyes, UV radiation, ethidium bromide, phenol, chloroform, high temperatures, extreme pHs, vortexing, or spin columns
         
      • Minimal introduction of nicks, gaps, and other "undetectable" single-stranded damage that may result in lower library prep yields and lower sequencing quality

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      PURITY REQUIREMENTS

      • DNA is purified and stored in EB or TE buffer; RNA is purified and stored in nuclease-free water 
      • Nanodrop 260/280 ratio = >1.8
      • Nanodrop 260/230 ratio = 2.0 - 2.2
      • No visible particulates, dust/lint, precipitates, colors, or cloudiness

      If your samples do not meet these purity requirements, GC3F can perform AMPure bead cleanup ($25 per sample) or Nanobind disc cleanup ($75 per sample), but this may drop the yield and/or quality and results are not guaranteed to be sufficient for PacBio prep. Thus it is recommended to perform any required cleanups prior to shipping your samples.

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      QUANTITY (YIELD) REQUIREMENTS

      • Quantify your nucleic acids 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.)
           
      • For genomic CLR libraries: Submit a minimum of 5 µg of dsDNA in EB or TE, but 10-20 µg is preferred (at preferred concentration >150 ng/µL). It is strongly recommend to use samples with >50% of the gDNA >30kb in length.
        • More input is always better and may allow us to "scale up" the prep reaction in order to select for the longest possible fragments to improve your genome assembly. Higher DNA input may also be required for higher number of SMRT cells requested. Pricing for scale-ups are noted in the table above.
        • If you have less than 5 µg of gDNA or are working with a small genome, please contact us to discuss options.
        • Multiplexing is not supported for CLR libraries because the insert length is too long to obtain sufficient subread passes of the barcode region in order to accurately call its sequence. This causes the demultiplexing pipeline to reject the vast majority of reads (can be >90%), resulting in very low data yield. 
           
      • For genomic HiFi libraries: Submit a minimum of 5 µg of dsDNA in EB or TE, but 10-20 µg is preferred (at preferred concentration >150 ng/µL). It is strongly recommend to use samples with >50% of the gDNA >30kb in length.
        • Depending on the number of SMRT cells requested, higher input may be required to "scale up" the prep reaction because the shearing, narrow size selection, and exonuclease chemistry required for HiFi will substantially drop yield. Pricing for scale-ups are noted in the table above.
        • We also offer the Low Input HiFi method (for <5 µg input) and the Ultra Low Input HiFi method (for <50 ng input), and also a combination of both methods if >1 SMRT cell is required. 
        • Multiplexing is supported via use of barcoded SMRT bell adapters.
           
      • For Iso-Seq libraries: Submit a minimum of 50 ng/µL of total RNA in at least 10 µL of nuclease-free water. It is strongly recommend to use samples with RIN/RQN >7.
        • If you have less than 500 ng total RNA, please contact us to discuss options.
        • If you are preparing your own cDNA from total RNA following PacBio's protocol, please submit 200-600ng of purified, multiplexed cDNA in EB.
        • Multiplexing is supported via use of barcoded cDNA primers and/or barcoded SMRT bell adapters.
           
      • For all other library types (amplicons, pulldowns, etc.): The minimum input required and other considerations will vary depending on the prep method used. Please contact us to discuss options!

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      SHIPPING GUIDELINES:

      Before sending any samples, please register for an iLab account and then click "Request Services" from the top menu bar in iLab to submit your requests for ALL associated services (HMW gDNA Extraction, Sample Prep, and/or PacBio Sequencing). It is important that all of your requests have been submitted in iLab before you provide the physical samples to us!

      After iLab submission, please deliver your samples to Tina Arredondo 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 Tina Arredondo 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!