With the addition of Oxford Nanpore’s GridION sequencer to our repertoire of third generation sequencing technologies, which include PacBio’s SMRT sequencing and 10X Genomics’ linked-read technology, UMN researchers are now able to directly sequence high-fidelity DNA or RNA molecules in real-time. Nanopore uses a revolutionary, propriety sequencing technology to pass single DNA molecules through a protein nanopore embedded in a polymer membrane, and as an ionic current is passed through the nanopore, the disruption in this current identifies the passing sequence of bases. When high-quality DNA (>100kb) or RNA is inputted through the pore, this technology is capable of producing ultra-long reads with the potential for 100’s of kb in length, ideal for improving genome assembly, resolving structural variants, transcriptome analysis, and metagenomic profiling.
As a small benchtop instrument with a straightforward library prep and loading protocol, the GridION is an optimal DIY instrument that gives users a rapid, scalable sequencing solution - ranging from one to five flow cells - that is capable of outputting up to 150 Gb in less than 48 hours when all five flow cells are used. The sample-to-analysis DIY workflow starts with high-quality DNA or RNA extractions that is meticulously assessed for quality and ends with MSI delivering FASTQ and BAM files for downstream analysis.
The UMGC provides GridION training on an as-requested-basis at our Snyder site on the St. Paul campus. A staff scientist, who specializes in long-read technologies, will guide new users through Flow Cell Check performance, instrument operation, and loading a flow cell. Training will be a hands-on experience on an actual library the user has prepared, in order to give users the ability to independently sequence their own future run. Providing space is available, up to two scientists from one lab can attend the two-day training (day one: 2 hours, day two: 1 hour) at a cost of $162.58/lab.
Reserving the GridION
After training, DIY users will be able to independently run a flow cell check and sequence their prepared library without further support from the UMGC staff. There is no fixed sequencing run time on the Nanopore and DIY users determine the sequencing run time based on their experimental goals and then reserve the instrument in one-day or two-day increments.
Matt Arriola at email@example.com will assist users in reserving the GridION for the requested amount of time to cover the flow cell check and sequencing run time at our Snyder site (available from 8:30 AM – 4:30 PM). Users must have a reservation in order to use to the GridION.
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, users 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.
High molecular weight (HMW) DNA typically cannot be achieved with standard extraction kits and methods and special pipetting attention and supplies are required to mitigate forces that cause DNA breakage. In addition, in order to prevent failure of the sensitive pores on the flow cell, the HMW DNA (or RNA) needs to be chemically pure with 260/280 and 260/230 between 1.8 and 2.2 as determined by NanoDrop.
To determine DNA and RNA quality and quantity, the UMGC recommends a thorough QC workup with fragment size analysis (Agilent TapeStation is sufficient but pulse-field gel electrophoresis is preferred for DNA), Nanodrop for absorbance ratios, and PicoGreen fluorescence for accurate quantification.
Alternatively, DIY users can elect to submit samples to our extraction service for intact, HMW DNA isolation, typically with sizes 50 kb or higher (depending on tissue type) as required for long-read sequencing, or for high-quality RNA isolation. Please contact our extraction team lead, Darrell Johnson, at firstname.lastname@example.org more information.
Purchasing reagents and flow cells
DIY users will purchase a GridION library prep kit directly through the Nanopore Store. A comprehensive range of kits are available that address low input, rapid prep, long reads and high-throughput requirements, and researchers will determine the appropriate kit based on their experimental design using the following as a selection guide: 1) sample type, 2) desired PCR conditions, and 3) whether multiplexing will be needed. Additional consumables are the responsibility of the researcher.
Up to five independent flow cells can be run simultaneously on the GridION X5 to process five different experiments or one experiment across all five for large-scale genome sequencing. Samples that have been barcoded using one of Nanopore’s barcoding kits can be run on the same flow cell. Run time is not fixed and each flow cell has a lifespan of ~48 hours with the potential to output 30 Gb of data, however, sequencing yield is ultimately determined by the quality of the input DNA.
Note: Flow cells are included in the DIY costs and researchers do not need to purchase flow cells through the Nanopore Store.
Final libraries prepared by the researcher are submitted to the UMGC for QC analysis, including size determination using Agilent’s ScreenTape analysis and quantification by PicoGreen. Libraries in sufficient amount and with a size distribution that corresponds to the library prep kit used will be recommended for sequencing, but ultimately, DIY users determine whether to move forward with sequencing. DIY users with passing libraries should then sign up for a flow cell check and reserve the GridION instrument for their sequencing run.
For additional questions on using the GridION, to set-up training, or to reserve the instrument, contact Matt Arriola at email@example.com.
|A staff scientist will guide new users on loading the GridION using an actual library. Up to two scientists from one lab can attend the training in Snyder Hall.||1||session||$162.58||$186.92|
|Client operated 24-hour run.|
|Client independently uses the GridION to fully execute their own sequencing run using a 24-hour run time. Rate includes flow cell and instrument usage.||1||day||$1,059.74||$1,219.77|
|Client operated 48-hour run.|
|Client independently uses the GridION to fully execute their own sequencing run using a 48-hour run time. Rate includes flow cell and instrument usage.||2||days||$1,134.25||$1,305.65|
How to sign-up
The UMGC provides training as needed in 28 Snyder Hall on the St. Paul campus. Training is scheduled through Matt Arriola at firstname.lastname@example.org and trainees supply a prepared library for a hands-on training experience.
Once GridION training is complete, users are able to reserve the GridION in one or two-day increments by emailing email@example.com. Please be sure your libraries have passed QC before reserving an instrument time. An EFS numbers will be needed to hold the instrument for the confirmed day.
Location for GridION training and operation
Lead: Matt Arriola
Operating Hours: 8:00 AM – 4:30 PM Monday – Friday
There are three options for transferring data from the UMGC to clients: delivery to the Minnesota Supercomputing Institute’s (MSI) high-performance file system, download from a secure website, or shipment on an external hard drive. Please indicate your data delivery preference when placing a order for sequencing.
Internal clients and external clients that opt-in to MSI storage have their data released to the client’s MSI group directory on the MSI high-performance filesystem (/home/GROUP/data_release/umgc), from where it can be analyzed using MSI’s high-performance computers or Galaxy system. The data is stored for five years in uncompressed format with read-only permissions to avoid accidental modification, and secure off-site tape backups of the data are maintained for disaster recovery. MSI will charge the client a storage fee for this service.
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.
External clients may have data shipped on a hard drive purchased by the UMGC and invoiced to the client.
MSI guarantees storage of client data for five years, after which the data can be transferred to tape for long-term storage, contact MSI (firstname.lastname@example.org) for more information.
Web download and hard drive
Data is typically recoverable for up to 5 years. If the external researcher should ever need the FASTQ files re-posted to the data download website or shipped on a hard drive within 5 years from data release, email email@example.com to initiate data recovery. The client will be charged for the cost of recovering the data from tape archive. The UMGC does not provide any guarantee that data can be successfully recovered from tape archive.