Nanopore GridION Sequencing

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. 

Applications:

• 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.

Output

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.

Benefits

• 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.

Extraction

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 extract@umn.edu for more information.

Questions

Please contact us at next-gen@umn.edu for project support using the ONT GridION or any of the sequencing technologies we offer at the UMN Genomics Center.