Our PacBio Revio sequencing service is live! Details below
Coming soon: PacBio Revio SPRQ Chemistry! Read more about it here!
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JUMP TO TOPIC:
- PacBio Services Overview
- PacBio Revio Overview
- Pricing Information
- Submission guidelines
- Submission Standards
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PacBio Services Overview
GC3F has provided PacBio sequencing since 2017 and has been a Certified Service Provider since 2021. With the acquisition of PacBio's Revio sequencer, we are now able to offer even more affordable and high-throughput sequencing.
We offer a variety of library prep options to support your HiFi sequencing needs. These include:
- Whole-genome SMRTbell libraries
- Kinnex full-length RNA libraries
- Concatenated amplicon sequencing (contact genomics@uoregon.edu for details)
We also offer sample prep services upstream of library prep, such as extraction of ultra-high molecular weight DNA, cDNA synthesis, or amplification of target genes. We are happy to work on "unconventional" projects and difficult-to-extract species.
Contact us at genomics@uoregon.edu to get started on your next SMRT Sequencing project or click here to submit a request through iLab.
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PacBio Revio
GC3F now offers sequencing on PacBio Revio. The Revio replaces our Sequel II and represents a significant increase in throughput, capable of running 16 SMRTcells per week and producing 30-90 Gb of High-Fidelity (HiFi) data per SMRTcell. Revio data automatically includes 5mC at CpG sites (methylation data) for native DNA.
For more details on the Revio system, visit PacBio's Revio informational page.
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PacBio Sequencing Prices:
Service | Internal Rate | External Rate |
PacBio Sample Prep Prices:
PacBio DNA library preps generate sufficient library for one Revio SMRT Cell. If your sample requires >1 SMRT Cell, we can run additional reactions at a reduced per-reaction rate. The same reduced per-reaction rate applies to multiple samples submitted concurrently for library prep, up to 8 reactions per batch.
Service | Internal Rate | External Rate |
$105 for 2nd-8th in batch | $155 for 2nd-8th in batch | |
Barcoded cDNA Synthesis for long-read sequencing | $200 per reaction | $300 per reaction |
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Submission Guidelines
iLab request IDs are used for tracking your samples. Submission of iLab requests before providing physical samples is critical to avoiding processing delays. Please register for an iLab account and then click "Request Services" from the top menu bar in iLab begin this process.
After iLab approval, samples can be dropped off in person or shipped frozen on dry ice. Shipment on dry ice is absolutely critical to successful sequencing. Please ensure that samples are extremely well sealed before shipping. Adhesive foil seals should be buffed on for extra security (video demonstration).
- Local users can bring samples to 288 Klamath Hall:
- Drop off DNA or completed libraries by placing tubes in the "PacBio Sequencing" box on shelf 1 of freezer 2.
- Frozen tissue must be handed off to lab personnel, who can place the sample directly in the -80.
- Non-frozen tissue must be flash-frozen prior to storage. Contact Tina Arredondo to coordinate LN2 availability at drop-off.
- Non-local users can ship samples frozen on dry ice in a well-protected vessel. Please ensure that your package will arrive at UO Mon-Fri (UO cannot accept deliveries on weekends or federal holidays). Packages can be mailed to the following address:
- University of Oregon/GC3F
- 1318 Franklin Blvd
- Room 273 Onyx Bridge
- Eugene, OR 97403
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Standards for Submission
Providing already-extracted DNA will result in significantly faster turnaround time than submitting tissue. If you need advice or resources for improving your HMW extractions to meet these standards, please reach out to Tina Arredondo at tarredon@uoregon.edu.
Tissue Requirements
- Freshly-harvested tissues should be flash-frozen in liquid nitrogen and stored at -80°C. Other preservation methods, such as RNAlater, typically result in poor sequencing performance.
- Amount:
- For plant extractions, provide at least 3-5g of flash-frozen young leaves
- For animal extractions, provide one of the following:
- >1g of low-DNA-content tissue (muscle, skin)
- >100mg of high-DNA-content tissue (liver, spleen)
- >50 ul of blood containing nucleated RBC
- >500 ul of blood containing non-nucleated RBC
- For cultured bacteria, >20M cells
- 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.
Nucleic Acid Quality
Samples must not have been exposed to intercalating dyes, UV radiation, ethidium bromide, high temperatures, extreme pHs, vortexing, or spin columns. These cause introduction of nicks, gaps, and other "undetectable" single-stranded damage that may result in lower library prep yields and lower sequencing quality. Additionally:
- DNA:
- Must be double-stranded (ssDNA cannot be prepped or sequenced)
- For WGS, >50% of the gDNA must be >30kb in length
- Samples have been treated with RNase and there is no remaining RNA present
- RNA:
- Strongly recommend to use samples with RIN/RQN >7
- Samples have been treated with DNase and there is no remaining DNA present
Nucleic Acid Purity
- 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 bead cleanup ($25 per sample), but results are not guaranteed to be sufficient for PacBio prep. Thus it is recommended to perform any required cleanups prior to shipping your samples.
Nucleic Acid Quantity
- Whole genome sequencing:
- A total of 3 µg of dsDNA per SMRTcell (5 µg is preferred)
- If multiplexing, >500 ng per sample and totaling at least 3 ug across samples to be multiplexed.
- For genomes <500 Mb and where obtaining 3 ug of DNA from a single individual is infeasible, we offer the Ultra Low Input HiFi method. This prep enables HiFi read generation from <50 ng of DNA, however uneven coverage from PCR and shorter read length (5-10kb) may make assembly more difficult.
- Kinnex full-length RNA libraries:
- A minimum of 50 ng/µL total RNA in at least 10 µL of nuclease-free water.
- If you plan to prepare your own cDNA, please contact us to coordinate downstream compatibility. Submit 200-600ng of purified, multiplexed cDNA in EB.
- For all other library types (amplicons, pulldowns, etc.): The minimum input required and other considerations will vary depending on the prep method used. Please email us at genomics@uoregon.edu to discuss options.
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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!