The UMGC continues to expand its long-read capabilities with the Oxford Nanopore Technologies (ONT) GridION X5 system.
Unlike other sequencing platforms that use optical detection, Nanopore devices feed template DNA through a membrane-bound protein nanopore across which an electrical voltage is applied. A docked motor protein translocates template DNA or RNA through the pore, and the fluctuations in ionic current are decoded by in silico basecalling.
Also unlike other platforms, Nanopore devices can also read RNA sequence directly, i.e. without the need for cDNA synthesis. Nanopore systems have no theoretical limit on the size of molecules that can be fed through the pore, meaning that mappable reads > 1Mbp are achievable.
These ultra-long reads span regions that are difficult to resolve using short-read sequencing and are able to produce highly contiguous genome assemblies, identify structural variants with substantially higher accuracy, and sequence tens to hundreds of thousands of full-length RNA transcripts in a single run. And since genomic DNA can be sequenced on nanopore devices without the need for fragmentation, amplification or strand synthesis, the long-range methylation information is retained in the data.
- Flexible sequencing of whole genomes, targeted regions, and full-length RNA transcripts
- Long reads enhance analysis of repetitive regions, structural variation, phasing, metagenomics, and more
- Quantify and characterize RNA splice variants, isoforms, and fusion transcripts
- Directly identify epigenetic modifications (e.g. methylation) alongside nucleotide sequence
The UMGC offers two types of nanopore sequencing services: 1) our “full” service that has the UMGC prepare the library and perform sequencing; or 2) researchers can arrange to be trained to operate the GridION through our Do-It-Yourself Genomics service for faster project turnaround.
Nanopore runs are tunable to the user’s specific needs. For example, a well-performing long amplicon run might yield sufficient data from only one flowcell in a matter of a few hours, allowing users to stop the experiment and retain flowcell sequencing capacity for later use (though users will be responsible for purchasing the entire flowcell).
At the other extreme, a large, polyploid plant genome sequencing experiment would likely use all five GridION X5 flowcell bays simultaneously--or even require multiple runs. One GridION flowcell can provide up to 72 hours of sequencing time, and multiplexing several samples on the same runs with barcodes is also possible depending on the specific workflow.
One GridION flowcell run for 72 h typically yields 5-15 Gbp of data, or ~2-5X human genome coverage.
Users have the option to receive either FAST5 (raw) and/or FASTQ (live basecalled) data from their GridION runs. As Nanopore’s basecalling algorithms continue to improve, retaining the raw data allows users to re-run basecalling at a later date, potentially improving the accuracy without having to run additional sequencing.
- Ultra-long Reads: With the potential for ultra-long read lengths up to 2 MB, investigators can assemble complex genomes and resolve repetitive regions that are difficult for short-read technologies.
- High Accuracy: a full suite of supported polishing tools routinely give consensus accuracy >QV40 (99.99%), with improvements to basecallers being rolled out regularly.
- Uniform Coverage: No bias based on GC content enables sequencing through regions inaccessible to other technologies. Template preparation and sequencing do not rely on amplification, thus there is no PCR bias for more uniform genome coverage.
- Epigenetics: With no PCR amplification step, base modifications are directly detected during sequencing without the need for bisulfite conversion.
- Direct RNA Sequencing: Allows the sequencing of native RNA molecules without fragmentation or amplification for unbiased, full-length transcript isoform sequences.
Like the other long-read technologies, Nanopore’s read length capability is dependent on the physical length and quality of the input DNA. To leverage the higher cost of using a long-read platform, researchers will want to maximize input DNA length (>100kb) in order to generate ultra-long reads with the potential for N50 read lengths between 20 – 40 kb, which is ideal for whole genome sequencing and discovering structural variations.
Researchers are encouraged to the UMGC’s extraction service for intact, HMW DNA isolation, that typically with sizes 50 kb or higher (depending on tissue type) as required for long-read sequencing, or for high-quality RNA isolation. Contact our extraction services at email@example.com for more information.
Please contact Dr. Jon Badalamenti at firstname.lastname@example.org for project support using the ONT GridION or any of the sequencing technologies we offer at the UMN Genomics Center.
How to Order
Please contact email@example.com for project specifications.
Samples can be dropped off at any of our campus locations
- 1-210 Cancer & Cardiovascular Research Building (Minneapolis campus)
- 20 Snyder Hall (St. Paul campus)
Please give advance notice of your sample submission date and time so staff can be prepared to receive samples. If shipping samples from outside the University of Minnesota, ship via express shipping carrier to the address below.
Please send the tracking information to firstname.lastname@example.org.
University of Minnesota Genomics Center
1475 Gortner Avenue
28 Snyder Hall
St. Paul, MN 55108
There are four options for transferring data from the UMGC to clients: 1) delivery to the Minnesota Supercomputing Institute’s (MSI) high-performance file system, 2) download from a secure website, 3) download with Globus, or 4) shipment on an external hard drive. Please indicate your data delivery preference when placing an order for sequencing.
1. MSI storage
Internal clients have their data released to MSI's Shared User Resource Facility Storage (SURFS). Delivered data will be located in the "data_delivery" folder in your group's folder on MSI's primary filesystem (home/GROUP/data_delivery/umgc). MSI does not charge for SURFS storage costs, but files expire and are removed one year after they've been delivered. Files should be copied to other MSI storage locations such as Tier2, Tier3, or your group's "shared" folder before they expire.
2. Web download
Internal clients that opt-out of MSI storage and external clients can download their data from a secure website using either a web browser or a command-line download tool, complete instructions are provided in an email from the UMGC. The client’s data is available for download for 30 days, after which the data will be removed from the data download website and the client takes responsibility for storing the data.
Internal and External clients can use Globus to download their data. This is the recommended method for external clients to download large datasets.
4. Hard drive
External clients may have data shipped on a hard drive purchased by the UMGC and invoiced to the client at a cost of $250 per hard drive.
The UMGC archives most customer data for a year and some datasets are retained for 5 years or more. If you need a dataset re-delivered email a request to email@example.com to initiate data recovery. The UMGC does not provide any guarantee that data can be successfully recovered from the archive.