Oxford Nanopore Technology
Oxford Nanopore uses a unique sequencing technology that allows the sequencing of nucleic acid molecules with lengths that range well above 100,000 bp, although typical genomic DNA samples produce read lengths with N50 values that can be as high as 40,000 bp. While genomic DNA is often sequenced on an Oxford Nanopore instrument, large PCR products, cDNA and RNA can also be sequenced. Furthermore, it is possible to determine the methylation of cytosines at CpG sites in genomic DNA directly without bisulfite treatment of input DNA.
Oxford Nanopore sequencing makes use of an engineered pore-forming protein that sits in a membrane. An electric charge is applied across the membrane, and ions flow through the pore resulting in an electrical current. When a single strand of DNA or RNA passes through the pore, the electrical current is altered according to the bases that are inside the pore. As the DNA or RNA continues through the pore, the current changes, and the sequence of the nucleic acid can be determined based on the observed current signal at a single pore. Because the length of sequence read does not depend on a polymerase, read lengths of dozens of kbp are common with genomic DNA. With high-quality input RNA, RNA and cDNA libraries can produce full length transcript reads. Yields for both DNA and RNA/cDNA sequencing are very dependent on sample quality. For DNA, yields of 50 Gbp (up to 70 Gbp) can be obtained with good quality DNA when sequenced on the PromethION flow cells. Yields of 10 Gbp (up to 20 Gbp) have been obtained on the GridION instrument. For transcript sequencing on the GridION, more than 10 million reads can be obtained with the cDNA-PCR sequencing kit, and several million reads can be obtained by the direct RNA and direct cDNA sequencing kits.
Sample Quality Requirements
Because Oxford Nanopore sequencing technology relies upon an engineered pore-forming protein, anything that adversely affects that pore can potentially disable a pore and stop a pore from generating sequence. A single flow cell contains up to 2,400 pores. The amount of sequence that is obtained from a sequencing run is largely dependent on the quality of the input DNA or RNA. For best results, submitted DNA should have a 260/280 ratio of 1.8 to 2.0 and a 260/230 ratio of 2.0 to 2.2. For RNA, the 260/280 ratio should be ~2.0, and the 260/230 ratio should be 2.0 to 2.2. DNA and RNA samples that do not meet these requirements likely contain contaminants that can adversely affect sequencing yields.
Additionally, for genomic DNA samples, it is essential that high molecular weight DNA is submitted for sequencing. Any indication of shearing in a gel image also suggests that there is extensive nicking in the sample. As Nanopore sequencing simply involves unwinding double-stranded DNA and passing a single strand through a protein pore, as soon as a nick is encountered, the single strand passing through the pore is broken, and only a short read will be obtained. The Genomics Core uses a protocol for genomic DNA libraries that includes a nick repair step, but extensive nicking cannot be completely fixed. Additionally, empirical evidence indicates that there is a size-bias of the DNA that enters the sequencing pore. Short DNA fragments are much more likely to be sequenced.
Additional sample requirements for various Oxford Nanopore libraries are described on our Nanopore Sample Requirements page.
Sample Preparation Methods
Because of the requirement for high-quality genomic DNA for Nanopore sequencing, many traditional purification methods (phenol-chloroform, guanidinium thiocyanate) will fail to produce acceptable DNA samples. Spin-column-based methods are generally discouraged as they can result in physically damaged DNA that will produce very short reads and low yields and should not be used for genomic DNA preparation. Methods that have been reported to work well can be dependent on the source of the sample. Qiagen Genomic-tip-based methods have worked well with animal tissue, blood samples, animal cell cultures, gram-negative bacterial cultures, gram-positive bacterial cultures. The Qiagen Blood and Cell Culture kits have worked with animal cell cultures, fish tissues and blood samples. The Qiagen DNeasy PowerMax Soil kit has produced high-quality DNA from soil and bacterial cell cultures. The Nanobind CBB DNA kit from PacBio works for animal cell cultures, blood samples and bacterial cultures. The Nanobind Plant Nuclei DNA kit has also been found to produce high-quality DNA from plants that will produce high yielding Nanopore sequencing runs. PacBio also has a Short Read Eliminator kit that can deplete shorter fragments from a DNA sample and increase the N50 of a long-read sequencing run. RevoluGen has a spin column-based kit that can reportedly isolate high molecular weight DNA from bacteria and animal cell samples.
High molecular weight (HMW) genomic DNA samples must be handled with great care. During isolation and all other handling conditions, HMW DNA must not be vortexed, and any pipetting must use wide bore tips. If wide bore pipette tips have not been purchased, it is acceptable to cut the tip off of a regular pipette tip with a new razor blade. The resulting pipette tip opening should be ~3 mm in diameter. All pipetting actions should be performed slowly. Sample mixing by repeated pipetting will shear the sample. Additionally, the use of low DNA binding microfuge tubes is recommended for HMW DNA.
Non-genomic DNA samples such as amplicons that are less than 20 kbp may be handled according to regular molecular biology practices, but samples that contain longer molecules should be handled with the precautions necessary to prevent physical shearing of the DNA.
For RNA samples, typical spin column protocols are acceptable for producing high-quality samples for Nanopore sequencing.
Sequencing Library Options
The MSU Genomics Core offers library preparations for the following types of samples.
|Library Kit Name||Kit ID||Sequencing Target|
|1D Ligation Sequencing Kit||
|1D Ligation Sequencing Kit with barcoding||LSK109+NBD104/
|Genomic DNA or Amplicons|
|Rapid Barcoding Kit||RBK004||Multiplexed Genomic DNA
|Direct RNA Sequencing Kit||RNA002||Full Length RNA|
|cDNA-PCR Sequencing Kit without barcodes||PCS109||Full Length cDNA|
|cDNA-PCR Sequencing Kit with barcoding||PCS109+PBK004||Multiplexing Full Length cDNA for a single flow cell|
Researchers who would like to prepare their own libraries for Nanopore sequencing can run those libraries in the MSU Genomics Core. In this case, the researcher will pay for the flow cell. However, because Nanopore libraries may not store well, it is necessary to coordinate with the Genomics Core before submitting any libraries to ensure that there are available slots on the GridION for running your libraries. Write to us at email@example.com so that we can discuss your sequencing needs.
Sequencing Results Files
The Genomics Core will return fast5 and fastq data files for all Nanopore sequencing runs. Fast5 files are Oxford Nanopore's version of the HDF5 file format. Fast5 files contain all of the raw signal data required to rebasecall your data if you choose to do that. The fastq files that are returned to you are the results of basecalling the fast5 files using the latest basecalling software from Nanopore. A summary of the all software that was used to produce your sequence results will also be provided.