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JUMP TO TOPIC:
- Oxford Nanopore Technologies (ONT) Sequencing Overview
- Pricing info
- Submission guidelines for user-prepped libraries
- Submission guidelines for input DNA/RNA for GC3F to prep your libraries on site
- Shipping guidelines
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GC3F IS NOW PROVIDING OXFORD NANOPORE TECHNOLOGIES (ONT) SEQUENCING
GC3F is pleased to further expand our capabilities in long-read sequencing with the addition of the Oxford Nanopore Technologies (ONT) sequencing platform to our service offerings. ONT sequencing provides many benefits that are unique to its technology of reading the electrical "squiggles" evoked across a membrane when nucleic acids are migrated through nanopores. The sequencing of native DNA molecules has many advantages. Completely free of PCR artifacts, and full of native methylation patterns that can be read for no additional cost of either DNA or money. The real-time base-calling of the ONT platform allows adaptive sampling, another free ONT benefit that can provide up to 10X enrichment for target genes or regions when doing whole genome sequencing. Full length cDNA libraries can be sequenced to give information on isoform and polyA length variation within RNAseq samples. Find out more of the possibilities at nanoporetech.com.
We offer both sample prep and sequencing for all currently available ONT kits. Sequencing is done using PromethION flow cells, and our instrument is the P2 Solo, which can run 2 PromethION flow cells concurrently. We can prep a variety of microbial, animal, plant, amplicon and any other samples and can work with the researcher to find the best balance between output (number of Gbp sequence yielded) and the average length of reads (N50). We also can do Ultra-Long sequencing, with reads 50 kb-1 MB+ possible. This Ultra-Long technology is optimized for mammalian samples, so other species may require pilot studies to investigate the feasibility of Ultra-Long sequencing.
Oxford Nanopore offers a number of unique advantages not found with any other platform. Two very important and useful features unique to ONT are native and FREE methylation data concurrent with regular DNA/RNA sequencing and the availability to select for or against specific nucleotide sequences (and even entire genomes) by utilizing the Adaptive Sampling technology. We can also align your sequence data to a reference while it is being sequenced so your finished result is an bam file pre-aligned to your provided reference.
Contact our team at genomics@uoregon.edu to find out more about all these new possibilities and to get started on your next ONT Sequencing project.
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ONT P2 SOLO INSTRUMENT
- Two independently controlled PromethION flow cells
- 24-72 hr run time depending on the application
- Any read length you need — from short amplicon to full-length cDNA (or direct mRNA sequencing!) to ultra-long sequencing (>4 Mb achieved)
- Read lengths are determined by your sample and experimental needs — no need to fragment your sample, making assembly, structural variant detection, and phasing easier
- Sequence DNA and RNA directly — meaning no amplification bias and retained modification information (e.g. methylation)
- ONT provided suite of analysis tools designed for accessibility to all scientists, not just bioinformaticians - Epi2Me
- Find out more about nanopore sequencing and how it works here!
CLICK HERE to submit a request for ONT library prep, sequencing, or HMW gDNA extraction! (Note, you will need to register for an iLab account.)
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ONT Sequencing Prices
Service |
Internal Rate |
External Rate |
|---|---|---|
| Oxford Nanopore PromethION Flowcell | $1,424.00 | $2,100.00 |
| Oxford Nanopore PromethION Flow Cell Wash & Reload | $50.00 | $75.00 |
ONT Sample Prep Prices
Service |
Internal Rate |
External Rate |
|---|---|---|
| Oxford Nanopore Ultra-Long gDNA Library Prep | $465.00 | $686.00 |
| Oxford Nanopore Ligation gDNA or full-length cDNA Sequencing Prep (1st sample per flow cell) | $465.00 | $686.00 |
| Oxford Nanopore Ligation DNA Sequencing Prep (additional samples) | $50.00 | $75.00 |
| Oxford Nanopore Rapid DNA Sequencing Prep (1st sample per flow cell) | $250.00 | $375.00 |
| Oxford Nanopore Rapid DNA Sequencing Prep (additional samples) | $50.00 | $75.00 |
| Oxford Nanopre Direct RNA Sequencing Prep | $399.00 | $588.00 |
| 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:
Only University of Oregon users may submit their own user-prepped libraries at this time, if this is something you are interested from outside UofO, please email genomics@uoregon.edu. UO users will need to submit the ONT 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 ONT 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 ONT library yield and high sequencing quality and quantity. 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
- See shipping guidelines below for further details
QUALITY REQUIREMENTS
- DNA/RNA fragments are of sufficient length for your desired sequencing application
- For gDNA, it is strongly recommend to use samples with >50% of the gDNA >15kb in length
- For cDNA or Direct RNA sequencing, it is strongly recommend to use samples with RIN/RQN >7
- No contaminating nucleic acids present
- For gDNA, samples have been treated with RNase and there is no remaining RNA present (please ensure that the RNAse has been removed by column or bead cleanup before submitting to us-active RNAse will wreak havoc on our QA/QC instrumentation!)
- For RNA, 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
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.
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 DNA libraries: Submit a minimum of 2 µg of dsDNA in EB or TE, but 3-5 µg is preferred (at concentration >12.5 ng/µL). It is strongly recommend to use samples with >50% of the gDNA >30kb in length.
- Depending on the number of PromethION flow cells requested, higher input may be required to generate sufficient library.
- We also offer DNA sequencing for samplesn with <50 ng of DNA, using PCR amplification, however uneven coverage from PCR and shorter read length may make assembly more difficult. In addition, methylation calls will not be possible as the DNA is not "native"
- Multiplexing is supported via use of barcoded ONT adapters
- For full-length cDNA 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.
- Multiplexing is supported via use of barcoded ONT 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 ONT 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 Jason Carriere 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.
University of Oregon/GC3F1318 Franklin BlvdRoom 273 Onyx BridgeEugene, OR 97403
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Please contact Jason Carriere for further details on ONT 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!