Nanopore sequencing technology provides the longest sequencing reads and, uniquely, direct RNA-sequencing. We offer multiple protocols for Oxford Nanopore Technologies (ONT) nanopore sequencing: Long-read and ultra-long-read DNA sequencing, targeted DNA sequencing, direct RNA sequencing, and full-length cDNA sequencing.
Please see the bottom of the page for an introduction to the technology and its other advantages.
Long-read DNA sequencing: This protocol is most suitable when high yields are desired. Sequencing adapters are ligated to the ends of long dsDNA molecules. The yields per flow cell can range from 50 Gb to > 100Gb depending on the input DNA quality, flow cell quality and the study organism. Sequencing read length N50s depend on the input DNA fragment lengths. Read length N50s of 30Kb -50Kb can be achieved when starting with high quality high molecular weight DNA. Sequencing run time is 72hr to 96 hrs. To improve the yield, the flow cell is typically washed 1-3 times during the run and fresh library is loaded after the wash.
Input DNA requirements – Long-read DNA sequencing preps best begin with 5 µg of total DNA as measured by fluorometric assay such as Qubit. The DNA should be RNA free. Nanodrop 260/280 nm ratio should be close to 1.8 and 260/230 ratio close to 2.0
For smaller genomes, up to 96 samples can be multiplexed on a single PromethION flow cell using PCR free barcoding.Ultra-long-read DNA sequencing: This protocol generates the longest sequencing reads including a significant proportion of reads with lengths longer than 100 Kb. It uses a transposase-based chemistry to attach sequencing adapters to DNA. The yields from ultra-long read libraries are lower than standard ligation libraries and can range from 20Gb to > 50Gb per PromethION flow cell depending on the input DNAquality and species.
Input requirements – We will perform DNA extractions in the core for ultra-long read sequencing. Please contact Ruta Sahasrabudhe ([email protected]) or Mohan Marimuthu ([email protected]) for further details.Full-length cDNA sequencing: Full-length cDNAs is prepared after priming transcripts from the poly-A tails (analogous to the Clontech SMART-seq protocol) and barcoded during PCR amplification. ONT adapters are ligated on the cDNA pool using rapid chemistry. These data are used for isoform analysis and gene annotations. 70 million – 100 million reads are generated per PromethION flow cell.
Direct RNA sequencing: Uniquely, the ONT platform can sequence RNA molecules in its native form without conversion to cDNA. This approach is especially suitable for studying RNA modifications. In this case the yield from one PromethION flow cell can range from 10 million to 20 million reads. Read length N50 ranges from 1.5 Kb – 2Kb depending on the input RNA quality.
Input RNA requirements – Full length cDNA or direct RNA sequencing requires 1µg of total RNA as measured by fluorometric assay such as Qubit in 10 µl nuclease free water or EB. RNA should be DNA free. Nanodrop 260/280 and 260/230 ratio should be 2.0. The RNA integrity scores (RIN scores) should be 8.0 or higher.
Sample requirements:
Genomic DNA samples: DNA samples have to be chemically pure with Nanodrop spectrometer 260/280 nm ratios between 1.8-2 and 260/230 nm ratios between 2.0-2.2. The DNA needs to be dissolved in 10 mM TRIS (pH=8.0-8.4) – e.g. Qiagen EB Buffer.
For long-read sequencing genomic DNA samples isolated with spin column protocols ( e.g. Qiagen DNeasy) are sufficient. Please submit at least 5µg of total DNA at a concentration of 100ng/µl in 50µl volume. Please submit a gel image.
For super-long-read sequencing we require high molecular weight DNA samples with fragment sizes over 50KB. Please submit at least 5µg of total DNA at a concentration of 100ng/µl in 50µl volume. Please submit a gel image.
For difficult DNA samples, especially plant DNA samples with hard-to-remove contaminants (e.g. some polysaccharides), we recommend to carry out a high-salt/phenol/chloroform cleanup . Please note that this protocol often leads to a loss of 50% of the sample. Before submitting DNA samples:
- Please email us a picture of an agarose gel of the sample. The run should contain also a marker of at least a 20 kb size (e.g. GeneRuler 1 kb Plus DNA Ladder or Lambda DNA/HindIII Digest Marker; suggested is a also a lane with undigested Lamdba phage DNA [48kb e.g. NEB N3011S]). Please run the electrophoresis slowly (e.g. at 80V depending on setup).
- Assess sample purity vie spectrophotometry - the 260/280 ratio should be between 1.8 to 2.0 and the 260/230 ratio should be higher than 2.0. PacBio recommends MoBio PowerClean columns for sample cleanup or the high-salt/phenol/chloroform protocol mentioned above if necessary.
- Please use fluorometric methods (e.g. Qubit) for DNA quantification if possible. Measure each sample at least 3 times and accept only reproducible measurements (HMW DNA is often not perfectly dissolved). Spectrophotometry is not reliable for quantifications (especially if the DNA extraction protocol used CTAB).
- DNA samples have to be RNA-free. The DNA isolation protocols should best include an RNAse digestion.
DNA isolation protocols provided on this PacBio page will generally produce suitable DNA samples for nanopore sequencing: Extractdnaforpacbio.com
Our lab offers HMW-DNA isolation as a service. Please see the sample requirements for DNA isolation page on this page.
RNA samples: For full-length cDNA sequencing and direct RNA sequencing we require 1 ug of total RNA in up to 20 ul of molecular-biology-grade water. The RNA samples need to be DNA-free and need to be accompanied by Bioanalyzer traces or equivalent; we can also perform this RNA integrity analysis for a fee. The samples should have RNA-integrity scores (RIN-scores) of 8 or higher.
Data delivery:
We will distribute Nanopore sequence data in fastq format via a secure server (Bioshare). The raw Nanopore sequence data in fast5 format are extremely large and unwieldy - each read is an individual file. We can distribute the raw data via hard drives if requested. We will transfer them to new portable hard drives and charge for the additional costs.
Advantages of Nanopore Sequencing Technology:
Nanopore sequencing with Oxford Nanopore Technologies (ONT) systems enables high-throughput long-read sequencing of both DNA and RNA samples as well as multiple base-modifications including 5mC and 5hmC. For highest molecular weight DNA (HMW-DNA) samples, read lengths of several hundred kb can be reached with ultra-long-read protocols. There is however a significant tradeoff between read lengths and expected sequence yield. Thus, frequently both, long-read and ultra-long-read, protocols are employed for genome assembly purposes. Nanopore sequencing data greatly enable de novo genome assemblies and structural genomic variant and transcriptome studies. High-quality genome assemblies are possible based on exclusively nanopore data. Read error correction with short-read data is no longer required.
ONT flow cells accurately measure the current/electrons that squeeze by single-stranded DNA molecule as it is ratcheted through a nanopore molecule between two compartments. The signal of the changing current is converted to base-calls via machine learning algorithms. To learn more about the technology, please see the ONT HOW-IT-WORKS page and the video below.
ONT graphic showing the Nanopore (blue) embedded in a membrane. The motor protein (green) sitting on top of the pore unwinds the double helix and ratchets a single DNA strand through the pore. The data plot illustrates the raw signals: measurements of the electric current passing through the pore. The strength of the current is influenced mostly by a stretch of five DNA bases located in the bigger cavity of the pore. Due to the ratcheting motion of the motor enzyme each base contributes to five signals.
We offer Nanopore sequencing on the highest throughput Nanopore sequencer, the PromethION. To our knowledge, we were the first academic core lab to offer such services. Depending on the samples and the library types, the PromethION can generate up to 100 Gb of sequence data per flow cell, and can run up to 24 flow cells simultaneously. We do offer HMW-DNA isolation as a service since high-quality single-molecule sequencing data depend on highest quality DNA samples. ONT has been progressing especially rapidly and is characterized by roughly annual flowcell chemistry changes and even more frequent basecaller software updates resulting in quickly improving sequence qualities.
The average sequence data yields can vary widely and will mostly depend on the sample properties (please see below). Average yields will be inversely correlated to the library molecule lengths (insert sizes). We have achieved the best run metrics mentioned below with DNA isolated from human/mammalian cell cultures and mammalian blood samples.
The sequence yields and run metrics will vary widely depending on the samples. As with other single-molecule sequencing technologies, the read lengths and the sequencing yields do depend on the nucleic acid sample quality. Every nick or chemical DNA adduct has the potential to abort reads and chemical contaminants could destroy the pores. The sample quality can be assessed in part by spectrometry and pulsed-field-gel-eletrophoresis (PFGE). We will QC the samples with both. Please note that these methods can not fully assess the quality and suitability of the DNA samples since they assess the DNA as double-stranded molecules. For example, single-strand nicks and chemical adducts could escape these methods. The QC data however allow us to rule out clearly problematic samples. It has been observed that genomic DNA samples from certain species (including some birds and Cnidaria) perform significantly worse than for example, mammalian samples. Thus it is possible that genomic DNAs of some species contain DNA base modifications that interfere with the current Nanopore protocols. As a consequence, we will perform test sequencing experiments (also on the smaller MinION sequencer) with new types of samples. Test sequencing is the only way to fully assess the suitability of DNA samples. We can't make any specific sequencing performance promises beforehand. It can be expected that Nanopore sequencing will generate on average longer reads as compared to sequencing on the Pacbio Revio. Please note, however, that Nanopore and PacBio data are not equivalent.
Our Nanopore sequencing expert is Ruta Sahasrabudhe, PhD. Please contact her at [email protected] for questions about HMW DNA extractions, Nanopore Sequencing & Hi-C projects.
In contrast to other sequencing technologies, the PromethION sequencer is mechanically very robust, since it is purely an electronic system and the chemistry is confined to the disposable flowcells. Thus, UC Davis scientists can get trained to sequence on the PromethION themselves during downtime periods.